The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p1-65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p-green fluorescent protein (GFP) or a Ure2p1-65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p1-65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] "crystal" of Ure2p poisons its propagation.     

Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae

We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to induce the appearance of the non-Mendelian factor [PSI]. This result was later interpreted by others as a crucial piece of evidence for a model postulating that [PSI] is a self-modified, prion-like conformational derivative of the Sup35 protein. Here we support this interpretation by proving that it is the overproduction of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of [PSI]. We also show that the "prion-inducing domain" of Sup35p is in the N-terminal region, which, like the "prion-inducing domain" of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein. This suggests that such a chimeric organization may be a common pattern of some prion elements. Finally, we find that [PSI] factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSI]-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.     

Enhanced expression of the yeast Ure2 protein in Escherichia coli: the effect of synonymous codon substitutions at a selected place in the gene

The expression of the yeast Ure2 protein and its two N- and C-terminal HA-(YPYPVDYA) epitope and His-tag fusions has been enhanced in E. coli by selected silent mutagenesis of the URE2 gene. The two Arg-AGA codons at positions 253 and 254 of the URE2 gene coding sequence were exchanged by CGT codons accordingly. This has allowed an increased yield (up to 100-fold) of the full-length protein synthesized. Western blotting with HA-epitope-specific antibodies using N- and C-terminal Ure2p-HA(epitope)-His-tag fusion constructs confirmed the integrity of the recombinant proteins. The N-(C-) terminal tagged proteins were shown to possess biological activity of the natural Ure2 protein.     

Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion curing

The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.     

The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities

The Ku heterodimer, conserved in a wide range of eukaryotes, plays a multiplicity of roles in yeast. First, binding of Ku, which is composed of a 70 kDa (Hdf1p) and an 80 kDa (Hdf2p) subunit [1-3], to double-strand breaks promotes non-homologous end-to-end joining of DNA [3]. Second, Ku appears to participate in DNA replication, regulating both the number of rounds of replication permissible within the cell cycle and the structure of the initiation complex [3,4]. Furthermore, mutations in HDF1 or HDF2 rapidly reduce telomeric poly (TG1-3) tract size [1-3], hinting also at a possible telomeric function of Ku. We show here that the two subunits of the Ku heterodimer play a key role in maintaining the integrity of telomere structure. Mutations in either Ku subunit increased the single-strandedness of the telomere in a cell-cycle-independent fashion, unlike wild-type cells which form 3' poly(TG1-3) overhangs exclusively in late S phase [5]. In addition, mutations enhanced the instability of elongated telomeres to degradation and recombination. Both Ku subunits genetically interacted with the putative single-stranded telomere-binding protein Cdc13p. We propose that Ku protects the telomere against nucleases and recombinases.     

DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258

The complexes of Hoechst 33258 with poly[d(A-T)2], poly[d(I-C)2], and poly[d(G-C)2], and poly[d(G-m5C)2] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)2] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)2] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45 degrees relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity--leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)2]. By contrast, when bound to poly[d(G-C)2] and poly[d(G-m5C)2], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)2] and poly[d(G-m5C)2] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed.     

Auxiliary downstream elements are required for efficient polyadenylation of mammalian pre-mRNAs

We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal. We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals. These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE. Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored. First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate. Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE. Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal. This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate. AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.     

Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.     

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), PtdIns(3)P, PtdIns(3,4,5)P3 and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to regulate GTPase activity in vitro [6] [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P2 is required for both early and late events in endocytic coated vesicle formation.     

Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells

Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.     

Lipid activation of CTP:phosphocholine cytidylyltransferase is regulated by the phosphorylated carboxyl-terminal domain

The role of the phosphorylated carboxyl-terminal domain of CTP:phosphocholine cytidylyltransferase (CT) in the regulation of enzyme activity was investigated by comparing the catalytic properties of wild-type CT to two mutant proteins with altered carboxyl-terminal phosphorylation domains. CT isolated from a baculovirus expression system was extensively phosphorylated at multiple sites in the carboxyl-terminal domain. The CT[S315A] mutant lacked a major CT phosphorylation site, and the carboxyl-terminal deletion mutant, CT[delta 312-367], was not phosphorylated. The higher activities of CT[delta 312-367] and CT[S315A] relative to CT were attributed to differences in the sensitivities of the enzymes to lipid activators. The rank order of the apparent Km values for activation by either phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol was CT > CT[S315A] > CT[delta 312-367]. In addition, CT exhibited negative cooperativity in its activation by phosphatidylcholine/oleic acid (nH = 0.64) and phosphatidylcholine/diacylglycerol (nH = 0.74) vesicles, whereas CT[delta 312-367] and CT[S315A] did not. These data support the concept that the phosphorylation of the CT carboxyl-terminal domain interferes with the activation of CT by lipid regulators.     

Phosphoinositide 3-kinase in rat liver nuclei

Biochemical and immunochemical data from the present investigation reveal the existence of a p85/p110 phosphoinositide 3-kinase (PI 3-kinase) in rat liver nuclei. 32P-Labeling of membrane phosphoinositides by incubating intact nuclei with [gamma-32P]ATP results in the formation of [32P]phosphatidyl-inositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3], accompanied by small quantities of [32P]phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies with subnuclear fractions indicate that the PI 3-kinase is not confined to nuclear membranes. The nuclear soluble fraction also contains PI 3-kinase and an array of inositide-metabolizing enzymes, including phospholipase C (PLC), phosphoinositide phosphatase, and diacylglycerol (DAG) kinase. As a result, exposure of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to the nuclear extract in the presence of [gamma-32P]ATP generates a series of 32P-labeled D-3 phosphoinositides and phosphatidic acid (PA) in an interdependent manner. On the basis of the immunological reactivity and kinetic behavior, the nuclear PI 3-kinase is analogous, if not identical, to PI 3-kinase alpha, and constitutes about 5% of the total PI 3-kinase in the cell. Moreover, we test the premise that nuclear PI 3-kinase may, in part, be regulated through the control of substrate availability by PtdIns(4,5)P2-binding proteins. Effect of CapG, a nuclear actin-regulatory protein, on PI 3-kinase activity is examined in view of its unique Ca2+-dependent PtdIns(4, 5)P2-binding capability. In vitro data show that the CapG-mediated inhibition of nuclear PI 3-kinase is prompted by PKC phosphorylation of CapG and elevated [Ca2+]. This CapG-dependent regulation provides a plausible link between nuclear PLC and PI 3-kinase pathways for cross-communications. Taken together, these findings provide definite data concerning the presence of an autonomous PI 3-kinase cycle in rat liver nuclei. The nuclear location of PI 3-kinase may lead to a better understanding regarding its functional role in transducing signals from the plasma membrane to the nucleus in response to diverse physiological stimuli.     

Maintenance and inheritance of yeast prions

The unusual genetic behaviour of two yeast extrachromosomal elements [PSI] and [URE3] is entirely consistent with a prion-like mechanism of inheritance involving an autocatalytic alteration in the conformation of a normal cellular protein. In the case of both yeast determinants the identity of the underlying cellular prion protein is known. The discovery that the molecular chaperone Hsp104 is essential for the establishment and maintenance of the [PSI] determinant provides an explanation for several aspects of the puzzling genetic behaviour of these determinants. What remains to be explained is whether these determinants represent 'disease states' of yeast or represent the first examples of a unique mechanism for producing a heritable change in phenotype without an underlying change in genotype.     

A role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-dependent cells

Activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) has been implicated in the induction of apoptosis in a variety of systems [1] [2] [3] [4] [5] [6] [7] [8]. BAF3 cells are pre-B cells that undergo apoptosis following IL-3 withdrawal or ceramide treatment [9] [10]. JNK/SAPK in BAF3 cells is stimulated by ceramide and also during cell proliferation in response to IL-3 [11], but its role in the apoptotic response is not clear. We have devised a method of selectively inhibiting JNK/SAPK activity using a dual-specificity threonine/tyrosine phosphatase, M3/6. Expression of this phosphatase in BAF3 cells prevented ceramide stimulation of JNK/SAPK activity but did not affect apoptosis following IL-3 withdrawal or ceramide treatment. IL-3-stimulated proliferation of BAF3 cells expressing the phosphatase was, however, inhibited. Hence JNK/SAPK activation is likely to be involved in the proliferative response of these cells but is not required for apoptosis. Selective ablation by dual-specificity phosphatases should be a general method for determining the functions of specific mitogen-activated kinase pathways.     

Efficacy of different schedules in the management of chronic hepatitis C with interferon-alpha

We have evaluated the kinetics of nitrogen dioxide production in a system for inhalation of nitric oxide. In addition to a small fraction of contamination of nitrogen dioxide in the nitric oxide stock gas, a considerable part of the total concentration of nitrogen dioxide is formed immediately after mixing of nitric oxide and oxygen. This initial build-up of nitrogen dioxide is followed by a linear, time-dependent increase in the concentration of nitrogen dioxide. An equation describing the concentration of nitrogen dioxide in the delivery system is formulated: [NO2] = kA x [NO] + kB x [NO]2 x [O2] + kC x t x [NO]2 x [O2], where nitrogen dioxide [NO2] and nitric oxide [NO] concentrations are in parts per million (ppm), oxygen concentration [O2] is expressed as a percentage and contact time (t) is in seconds. The rate constants are kA = 5.12 x 10(-3), kB = 1.41 x 10(-6) and kC = 0.86 x 10(-6). Calculated nitrogen dioxide values correlated well with measured concentrations. This new finding of an initial build-up of nitrogen dioxide has to be taken into consideration if the conversion of nitric oxide to nitrogen dioxide is to be calculated and in the safety guidelines for the use of nitric oxide.     

Three-dimensional structure of meso-diaminopimelic acid dehydrogenase from Corynebacterium glutamicum

Diaminopimelate dehydrogenase catalyzes the NADPH-dependent reduction of ammonia and L-2-amino-6-ketopimelate to form meso-diaminopimelate, the direct precursor of L-lysine in the bacterial lysine biosynthetic pathway. Since mammals lack this metabolic pathway inhibitors of enzymes in this pathway may be useful as antibiotics or herbicides. Diaminopimelate dehydrogenase catalyzes the only oxidative deamination of an amino acid of D configuration and must additionally distinguish between two chiral amino acid centers on the same symmetric substrate. The Corynebacterium glutamicum enzyme has been cloned, expressed in Escherichia coli, and purified to homogeneity using standard biochemical procedures [Reddy, S. G., Scapin, G., & Blanchard, J. S. (1996) Proteins: Structure, Funct. Genet. 25, 514-516]. The three-dimensional structure of the binary complex of diaminopimelate dehydrogenase with NADP+ has been solved using multiple isomorphous replacement procedures and noncrystallographic symmetry averaging. The resulting model has been refined against 2.2 A diffraction data to a conventional crystallographic R-factor of 17.0%. Diaminopimelate dehydrogenase is a homodimer of structurally not identical subunits. Each subunit is composed of three domains. The N-terminal domain contains a modified dinucleotide binding domain, or Rossman fold (six central beta-strands in a 213456 topology surrounded by five alpha-helices). The second domain contains two alpha-helices and three beta-strands. This domain is referred to as the dimerization domain, since it is involved in forming the monomer--monomer interface of the dimer. The third or C-terminal domain is composed of six beta-strands and five alpha-helices. The relative position of the N- and C-terminal domain in the two monomers is different, defining an open and a closed conformation that may represent the enzyme's binding and active state, respectively. In both monomers the nucleotide is bound in an extended conformation across the C-terminal portion of the beta-sheet of the Rossman fold, with its C4 facing the C-terminal domain. In the closed conformer two molecules of acetate have been refined in this region, and we postulate that they define the DAP binding site. The structure of diaminopimelate dehydrogenase shows interesting similarities to the structure of glutamate dehydrogenase [Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., & Stillmann, T.J. (1992a) J. Mol. Biol. 228, 662-671] and leucine dehydrogenase [Baker, P.J., Turnbull, A.P., Sedelnikova, S.E., Stillman, T. J., & Rice, D. W. (1995) Structure 3, 693-705] and also resembles the structure of dihydrodipicolinate reductase [Scapin, G., Blanchard, J. S., & Sacchettini, J. C. (1995) Biochemistry 34, 3502-3512], the enzyme immediately preceding it in the diaminopimelic acid/lysine biosynthetic pathway.     

Enzyme defects in glutamate-requiring strains of Schizosaccharomyces pombe

Among the glutamate-requiring strains of Schizosaccharomyces pombe previously described [1], glu2 and glu3 strains were both shown to lack NAD-specific isocitrate dehydrogenase. glu4 strains were shown to lack glutamine:2-oxoglutarate aminotransferase (GOGAT), and to be defective in ammonia assimilation. The regulation of GOGAT activity in wild-type cells was investigated and was consistent with GOGAT and glutamine synthetase being involved in ammonium assimilation, particularly under conditions of nitrogen limitation.     

The effect of transport-blocking drugs on secretion of fluid and electrolytes by the mandibular gland of red kangaroos, Macropus rufus

Mechanisms of primary fluid formation by macropodine mandibular glands were investigated in anaesthetized red kangaroos using ion-transport and carbonic anhydrase inhibitors. Bumetanide at carotid plasma concentrations of 0.005-0.1 mmol/l progressively reduced a stable, acetylcholine-evoked flow rate of 1.02 +/- 0.024 ml/min to 0.16 +/- 0.016 ml/min (mean +/- SEM). Concurrently, saliva [Na], [Cl] and osmolality decreased, [K] and [HCO3] increased and HCO3 excretion was unaffected. High-rate cholinergic stimulation was unable to increase salivary flow above 12 +/- 1.5% of that for equivalent pre-bumetanide stimulation. Furosemide (1.0 mmol/l) and ethacrynate (0.5 mmol/l) caused depression of salivary flow and qualitatively similar effects on ion concentrations to those of bumetanide. Amiloride (up to 0.5 mmol/l) caused no reduction in salivary flow rates or [Na] but decreased [K] and [Cl] and increased [HCO3]. When compared with bumetanide alone, amiloride combined with bumetanide further augmented [K] and [HCO3] and lowered [Cl], but had no additional effects on Na or flow. At the higher level, 4-acetamido-4'- isothiocyanatostilbene-2,2'disulphonic acid (SITS) (0.05 and 0.5 mmol/l) stimulated fluid output, increased [HCO3] and [protein], and depressed [Na], [K] and [Cl]. Relative to bumetanide alone, SITS given with bumetanide had no additional effects on salivary flow or electrolytes. Methazolamide (0.5 mmol/l) in combination with bumetanide curtailed the decrease in [Cl] and the increases in [K] and [HCO3] associated with bumetanide. The residual methazolamide-resistant HCO3 excretion was sufficient to support 2-6% of primary fluid secretion. It was concluded that secretion of primary fluid by the kangaroo mandibular gland is initiated mainly (> 90%) by Cl transport resulting from Na-K-2Cl symport activity. A small proportion of the fluid secretion (up to 6%) appears to be supported by HCO3 secretion. No evidence was found for fluid secretion being dependent on Cl transport involving Na/H and Cl/HCO3 antiports or on HCO3 synthesis involving carbonic anhydrase.     

Sertraline for premenstrual dysphoric disorder

Symptomatic cryoglobulinaemia is infrequent in HIV-1-infected patients, but a few cases have been described [1-3], occasionally associated with hepatitis C virus (HCV) infection [1, 3]. These cases showed rheumatologic [1] or neurologic manifestations [2, 3], but until now no cutaneous symptoms associated with cryoglobulinaemia in HIV-infected patients have been described. We report what we believe to be the first case of cutaneous, symptomatic cryoglobulinaemia in an HIV-1-positive patient, who, in addition, was HCV-negative.