A closer look at 30 day hospital readmissions after head and neck cancer surgery.

PURPOSE: 30-day hospital readmissions after head and neck cancer surgery continue to be a significant source of patient harm and healthcare expenditure. While there is substantial data in the literature assessing predictive factors for readmissions after head and neck cancer surgery, there are a paucity of studies which attempt to understand if such readmissions are preventable. The goal of this paper is to determine factors associated with 30-day hospital readmissions after head and neck cancer surgery and to understand if these readmissions were preventable. MATERIALS AND METHODS: Retrospective review from a single academic tertiary care center. Patients readmitted within 30 days after undergoing surgery for cancers of the head and neck between 2015 and 2018 were identified. RESULTS: Over a 3-year period, 26 patients undergoing resection with or without reconstruction of head and neck cancers were readmitted to the hospital within 30 days of discharge. There were 15 (58%) men and 11 (42%) women with a mean age of 68 years (SD 14 years). Twenty-one (81%) patients had squamous cell carcinoma and 13 (50%) had a primary site in the oral cavity. Thirteen (50%) had undergone free or regional flap reconstruction. The indication for readmission was related to the surgical wound in 19 (73%) and to medical complications in 7 (27%). Each case was categorized as "possibly preventable" versus "uncertain if preventable" based on whether a reasonable and feasible change in management may have prevented readmission. Six (23%) readmissions were deemed possibly preventable. Four were related to the surgical wound where initial free or regional flaps may have prevented complication. Two were medical complications that may have benefited from longer inpatient observation. CONCLUSIONS: For a subset of patients readmitted within 30 days of head and neck cancer surgery, a reasonable and feasible change in management may have prevented their hospital readmission. The significance of better understanding this patient population is underscored by the high mortality rate.

The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus.

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

Use of recombinant gp43 isoforms expressed in Pichia pastoris for diagnosis of paracoccidioidomycosis.

gp43 is the main diagnostic antigen for paracoccidioidomycosis (PCM). In vitro, gp43 expression in supernatant fluids of Paracoccidioides brasiliensis cultures can be unstable, and its regulation is poorly understood. We have been able to express soluble recombinant gp43 (gp43r) isoforms as N-mannosylated proteins secreted in the supernatants of Pichia pastoris cultures induced with methanol. They were secreted as major components from day 2 of induction and could be purified with affinity columns containing anti-gp43 monoclonal antibodies. We have expressed P. brasiliensis GP43 (PbGP43) sequences from genotypes A, D, and E, and the correspondent gp43r isoforms (gp43r A, -B, and -C, respectively; 200 ng) were compared to native gp43 in immunodiffusion (ID) and dot blot assays. Among 90 PCM patient sera showing ID-positive reactions with purified native gp43, 100% were positive with gp43rD and gp43rE and 98% reacted with gp43rA. Of these sera, 78 were tested in dot blot assays at a 1:1,000 dilution, and 100% reacted with all recombinant isoforms. In ID assays, the specificity was 100%, since 40 sera from patients with related mycoses and 30 sera from healthy individuals did not react with any of the antigens. In dot blot assays, 100% specificity for PCM occurred when cross-reactive mannose epitopes were neutralized with 10 mM metaperiodate or eliminated through deglycosylation. However, a 1:1,000 serum dilution was already discriminatory for most sera. We suggest that P. pastoris recombinant gp43, especially isoforms D and E, may replace the native antigen in ID and dot blot assays for diagnosis and prognosis of PCM. Regulated expression of large amounts of antigen in nonpathogenic yeast would justify its preferred use.

Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37 degrees C and 26 degrees C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (alpha-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.

Cloning and characterization of a LON gene homologue from the human pathogen Paracoccidioides brasiliensis.

A LON gene homologue from the human pathogen Paracoccidioides brasiliensis (PbLON) has been cloned, sequenced and characterized. It encodes a putative ATP-dependent proteinase Lon, which in Saccharomyces cerevisisae (PIM1) is a heat-inducible protein involved in the degradation of abnormal or short-lived proteins in the mitochondria. The PbLON ORF is within a 3369 bp fragment interrupted by two introns located in the 3'segment. The 5' and 3' regions flanking the ORF contain sequences which resemble known transcription elements. Several transcription binding factor motifs have also been found, including sites for heat shock/stress response and nitrogen control. The deduced protein consists of 1063 residues containing a mitochondrial import signal at the N-terminus and conserved ATP-binding (GPPGVGKT) and serine catalytic (KDGPSAG) sites. It shares high identity with Lon homologues from S. cerevisiae (73%), Homo sapiens (62%) and Escherichia coli (56%). In P. brasiliensis, an MDJ1 putative gene has also been partially sequenced adjacent to PbLON, possibly sharing divergently orientated promoter elements. This chromosomal organization is interesting, since Mdj1p is a heat shock chaperone essential for substrate degradation by PIM1 in yeast.

Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis.

The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.

DNA-based vaccination against murine paracoccidioidomycosis using the gp43 gene from paracoccidioides brasiliensis.

Gp43, the major 43-kDa antigenic glycoprotein of Paracoccidioides brasiliensis, or its 15-amino acid inner peptide (P10), induces a T-CD4(+), Th1 cellular immune response which protects BALB/c mice from intratracheal infection by virulent yeast forms. We investigated whether DNA vaccination using the gp43 gene could elicit protective immunity against P. brasiliensis. Animals immunised intramuscularly (i.m.) or intradermally (i.d.) with plasmid DNA containing the gp43 gene induced a specific, long lasting humoral and cellular immune response. A mixed Th1/Th2 cellular immune response in DNA-immunized mice was modulated in vivo by IFN-gamma and was protective in BALB/c mice. A significant decrease in the lung colony forming units (CFUs) and reduced, or no dissemination to the spleen and liver of immunised mice were observed.

Fungal morphogenesis and virulence.

Phenotypic variability in pathogenic fungi has long been correlated with virulence, but specific genetic and molecular mechanisms are only recently being unraveled. Fungal morphogenesis, reflecting the expression of several regulated genes, and the capacity of the rising forms or phases to cause disease has been focused on at the XIVth Congress of the International Society for Human and Animal Mycology. Three experimental models of pathogenic fungi have been discussed. In Cryptococcus neoformans, phenotypic variability or switching represents controlled and programmed changes rather than random mutations. Evaluated phenotypic traits were the capsular polysaccharide, cell and colony morphology and virulence. In the dimorphic Paracoccidioides brasiliensis, the serine-thiol proteinase from the yeast phase cleaves the main components of the basal membrane, thus being potentially relevant in fungal dissemination. In Candida albicans, relationships between adhesion proteins and those of lymphocytes and neutrophils are related to fungal pathogenicity. Regulation of the directional growth of hyphae and its tropic responses are correlated with the invasive potential of C. albicans.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp.

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37 degrees C, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme

Mapping of the T-cell epitope in the major 43-kilodalton glycoprotein of Paracoccidioides brasiliensis which induces a Th-1 response protective against fungal infection in BALB/c mice.

The 43-kDa glycoprotein of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis, the prevalent systemic mycosis of Latin America. Apart from eliciting high antibody titers, gp43 is also immunodominant in delayed-type hypersensitivity reactions in infected animals and humans. The cellular immune response in mice to gp43 administered in complete Freund's adjuvant involves CD4+ Th-1 lymphocytes, secreting gamma interferon (IFN-gamma) and interleukin 2 (IL-2) but not IL-4 and IL-10. The T-cell epitope of this antigen was mapped to a 15-amino-acid peptide (P10) based on lymphoproliferations with primed cells from three different haplotypes and on a computer-assisted protein analysis. The structural requirements of the T-cell epitope were determined by assaying a series of P10 analogous and truncated peptides. Only 12-mer or longer sequences were active, confirming presentation by major histocompatibility complex II. The HTLAIR inner core of P10 is the essential domain of the epitope, with various flanking regions possible. Immunization of mice with both gp43 and P10 led to vigorous protection against intratracheal challenge by virulent P. brasiliensis, with a >200-fold decrease in lung CFU and halting of dissemination to the spleen and liver. The protective effect of P10 is mainly attributed to an IFN-gamma-mediated cellular immune response. Unlike gp43, which induces an antibody response compatible with both Th-1 and Th-2 activation in infected BALB/c mice, P10 does not induce a humoral response. Protection by gp43 and P10 was characterized by a few well-demarcated lung granulomas with numerous nonviable yeast forms or resolved lesions with no detectable fungal cells.

Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane.

We have previously characterized an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis, using as substrates peptides analogous of the internally quenched fluorogenic peptide Abz-MKRLTL-EDDnp. In this communication, detection of maximal proteinase activity in the culture supernatant fluids followed the abrupt increase in the medium pH, owing to the accumulation of ammonia generated by urease activity. Culture supernatant fluids collected at the peak of proteinase activity against Abz-MRKLTL-EDDnp were able to cleave components of the basal membrane of the extracellular matrix (EM), including laminin, fibronectin, collagen type IV and proteoglycans, and the proteolytic activity was selectively inhibited both by PMSF and p-HMB (sodium 7-hydroxymercuribenzoate), which are also specific inhibitors of the serine-thiol proteinase. Human collagen I, bovine fibrinogen, human immunoglobulin G, BSA or P. brasiliensis gp43 were resistant to proteolysis. The kinetics of appearance of the proteinase activity against EM substrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp. Moreover, chromatographic fractions of culture supernatants containing the serine-thiol proteinase at high specific activity were also active against EM substrates. These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the first step for fungal tissue invasion.

Monoclonal antibodies against the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis.

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin. Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction. Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells. Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity. Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis.

The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis.

An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.

Biochemistry and molecular biology of the main diagnostic antigen of Paracoccidioides brasiliensis.

The 43,000 dalton glycoprotein of Paracoccidioides brasiliensis (gp 43) is the main exocellular antigen recognized by sera from patients with paracoccidioidomycosis in a variety of serological assays. Specific conformational peptide epitopes are recognized by the human antibodies as determined by antigen deglycosylation. Procedures for the purification of the gp43 using immunoaffinity chromatography have been described. The secretion of the gp43 as a function of the growth curve, its partial aggregation with a proteolytic enzyme, ability to bind laminin, as well as to form circulating immunocomplexes in vivo could play a role in pathogenesis. Crude antigenic preparations depleted of gp43 epitopes lost their ability to elicit positive skin tests. Accordingly, the purified gp43 molecule induced delayed hypersensitivity reactions in man and infected animals, caused a T-CD4-dependent proliferation of lymph node cells from mice immunized with it, and of peripheral blood lymphocytes from an individual sensitized to P. brasiliensis by prolonged contact with the fungus. To identify the immunodominant epitopes in both humoral and cellular reactions, the gp43 gene has been cloned, sequenced, and partly expressed. It bears peptide sequences homologous to those of beta-1,3-glucanases from Candida albicans and Saccharomyces cerevisiae but has no enzymatic activity itself. The molecular weight of the unglycosylated antigen is 42,227. A single N-linked oligosaccharide chain in the gp43 contains alpha-D-mannopyranosyl, beta-D-galactofuranosyl and N-acetylglucosaminyl units with the predominant ratio of 10:2:2, and characteristics of a high mannose type.

Disruption of the processing alpha-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae.

Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis.

Yeast KRE2 defines a new gene family encoding probable secretory proteins, and is required for the correct N-glycosylation of proteins.

We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.