Phylloporus and Phylloboletellus are no longer alone: Phylloporopsis gen. nov. (Boletaceae), a new smooth-spored lamellate genus to accommodate the American species Phylloporus boletinoides.

The monotypic genus Phylloporopsis is described as new to science based on Phylloporus boletinoides. This species occurs widely in eastern North America and Central America. It is reported for the first time from a neotropical montane pine woodland in the Dominican Republic. The confirmation of this newly recognised monophyletic genus is supported and molecularly confirmed by phylogenetic inference based on multiple loci (ITS, 28S, TEF1-α, and RPB1). A detailed morphological description of P. boletinoides from the Dominican Republic and Florida (USA) is provided along with colour images of fresh basidiomata in habitat, line drawings of the main anatomical features, transmitted light microscopic images of anatomical features and scanning electron microscope images of basidiospores. The taxonomic placement, ecological requirements and distribution patterns of P. boletinoides are reviewed and the relationships with phylogenetically related or morphologically similar lamellate and boletoid taxa such as Phylloporus, Phylloboletellus, Phyllobolites and Bothia are discussed.

Senior house officers and foundation year doctors in emergency medicine: do they perform equally? A prospective observational study.

INTRODUCTION: Implementing foundation and specialty training programmes within emergency medicine raised concerns about the potential work productivity and effectiveness of new junior doctors. Between August 2006 and July 2007 senior house officers (SHO) on 6-month posts and foundation year 2 (FY2) doctors on 4-month placements worked on the same roster, rotating between the emergency department at Ninewells Hospital, a university teaching hospital in Dundee, and a smaller affiliated unit at Perth Royal Infirmary. To compare the efficiency and productivity of both groups of junior medical staff. METHODS: A prospective observational study was performed at both departments using the number of patients seen per hour as an indicator of productivity. These rates were calculated using information gathered from a computerised patient record and management system. Analysis was performed using unpaired t tests. RESULTS: Both groups demonstrated a significant rise in performance between the first and last month of their attachment. There was no statistical performance difference between months 4 and 6 of the SHO group, and no significant statistical difference existed between the two groups over the study period. CONCLUSIONS: With FY2 trainees changing every 4 months, departments are potentially exposed to reduced productivity particularly in month 1. Whereas FY2 trainees have no performance difference when compared with their peers, their presence has undoubtedly impacted on middle and senior staff. Only 65% of patients attending this department are seen by junior medical staff and the vast majority of these are reviewed by senior doctors. Increasing supervision, teaching and assessments improve training, but has reduced shop floor presence and productivity.

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.

Determinants of human apolipoprotein [a] secretion from mouse hepatocyte cultures.

Efforts to develop an in vitro model system to analyze apolipoprotein [a] (apo[a]) gene transcription, mRNA translation, and protein secretion have been complicated by the limited tissue and species distribution of apo[a] and the presence of regulatory DNA sequences remote from the apo[a] transcription start site. In the current study we examined primary hepatocytes cultured from apo[a] transgenic mice as a model system for analyzing apo[a] biogenesis. Hepatocytes from mice transgenic for a yeast artificial chromosome (YAC) encoding the entire apo[a] gene in its own genomic context (YAC-apo[a] hepatocytes) were unable to maintain apo[a] expression beyond 48 h of culture. This suggests that the apo[a] promoter was not active in cultured YAC-apo[a] hepatocytes. In contrast, apo[a] expression was maintained for at least 7 days in hepatocytes cultured from mice transgenic for an apo[a] cDNA under control of the mouse transferrin promoter (transferrin-apo[a] hepatocytes). Pulse-chase experiments established that more than 80% of apo[a] synthesized by both transferrin-apo[a] and YAC-apo[a] hepatocytes was degraded prior to secretion, independently of the coexpression of human apoB.Thus, low secretion efficiency appears to be a general characteristic of human apo[a] proteins in mouse liver. Apo[a] secretion was increased somewhat (from 18% to 32%) in the presence of lipoprotein-containing serum. Transformed cell lines derived from transferrin apo[a] hepatocytes retained characteristics of apo[a] secretion similar to those observed in primary cells. Primary and transformed apo[a] transgenic hepatocytes may provide valuable additional models with which to study posttranslational mechanisms regulating apo[a] secretion. - Wang, J., J. Boedeker, H. H. Hobbs, and A. L. White. Determinants of human apolipoprotein [a] secretion from mouse hepatocyte cultures. J. Lipid Res. 2001. 42: 60;-69.

Role of calnexin, calreticulin, and endoplasmic reticulum mannosidase I in apolipoprotein(a) intracellular targeting.

Apolipoprotein(a) [apo(a)] is a component of atherogenic lipoprotein(a) [Lp(a)]. Differences in the extent of endoplasmic reticulum (ER) associated degradation (ERAD) of apo(a) allelic variants contribute to the >1000-fold variation in plasma Lp(a) levels. Using human apo(a) transgenic mouse hepatocytes, we analyzed the role of the ER chaperones calnexin (CNX) and calreticulin (CRT), and ER mannosidase I in apo(a) intracellular targeting. Co-immunoprecipitation and pulse-chase analyses revealed similar kinetics of apo(a) interaction with CNX and CRT, peaking 15-30 min after apo(a) synthesis. Trapping of apo(a) N-linked glycans in their monoglucosylated form, by posttranslational inhibition of ER glucosidase activity with castanospermine (CST), enhanced apo(a)-CNX/CRT interaction and prevented both apo(a) secretion and ERAD. Delay of CST addition until 20 or 30 min after apo(a) synthesis [when no apo(a) had yet undergone degradation or Golgi-specific carbohydrate modification] allowed a portion of apo(a) to be secreted or degraded. These results are consistent with a transient apo(a)-CNX/CRT association and suggest that events downstream of CNX/CRT interaction determine apo(a) intracellular targeting. Inhibition of ER mannosidase I with deoxymannojirimycin or kifunensine had no effect on apo(a) secretion, but inhibited proteasome-mediated apo(a) ERAD even under conditions where apo(a)-CNX/CRT interaction was prevented. These results suggest a role for an additional, mannose-specific, ER lectin in targeting secretory proteins to the proteasome for destruction.

Acute laryngeal oedema and the yellow nail syndrome.

A complication of yellow nail syndrome after pleurodesis is presented. After endotracheal extubation the patient developed delayed acute laryngeal oedema requiring re-intubation. We postulate that the combination of lymphatic defect and airway trauma from the double lumen endotracheal tube were contributory to the oedema. If endotracheal intubation is performed, these patients should be observed for 48 h.

Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion.

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of approximately 65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST. These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity.

The epidemiology of women's rugby injuries.

OBJECTIVE: The purpose of this study was to identify all injuries to members of an elite women's rugby team and to compare these injuries with published data on injuries in other women's contact and collision sports. DESIGN: This was a prospective cohort observational study conducted using a monthly log completed by the team's certified athletic therapist to closely monitor attendance at practices and games along with the type and severity of injuries. SETTING: Rugby games and practices held in Ontario, Quebec, and the Netherlands. PARTICIPANTS: Forty members of the Ontario Women's Senior Provincial Rugby Team over the 1997 season and the 1998 World Championships. MAIN OUTCOME MEASURES: An injury was defined as a rugby-related event that kept a player out of practice or competition for >24 hours or required the attention of a physician (e.g., suturing lacerations) and in addition included all dental, eye, and nerve injuries and concussions. RESULTS: There were a total of 35 injuries in 4,958 player-hours and 2,926 athletic exposures. This resulted in a rugby injury rate of 7.1+/-0.4 per 1,000 player-hours and 12.0+/-2 per 1,000 athletic exposures. CONCLUSION: The incidence of injuries in women's rugby is comparable with that in other women's contact and collision sports, indicating that the sport may be safer than stated in the literature and media.

Lipoprotein(a): intrigues and insights.

Lipoprotein(a) is an atherogenic, cholesterol ester-rich lipoprotein of unknown physiological function. The unusual species distribution of lipoprotein(a) and the extreme polymorphic nature of its distinguishing apolipoprotein component, apolipoprotein(a), have provided unique challenges for the investigation of its biochemistry, genetics, metabolism and atherogenicity. Some fundamental questions regarding this enigmatic lipoprotein have escaped elucidation, as will be highlighted in this review.

6-Aminohexanoic acid as a chemical chaperone for apolipoprotein(a).

Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25-200 mM) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8-14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).

Presecretory degradation of apolipoprotein [a] is mediated by the proteasome pathway.

Plasma levels of atherogenic lipoprotein [a] (Lp[a]) vary over a 1000-fold range and are largely determined by the gene for its unique glycoprotein, apolipoprotein [a] (apo[a]). The apo[a] locus comprises more than 100 alleles, encoding proteins from <300 to >800 kDa. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the secretion efficiency of apo[a] allelic variants contribute to the variation in plasma Lp[a] levels. In the current study, we investigated the mechanism of apo[a] presecretory degradation. The proteasome inhibitors, acetyl-leucyl-leucyl-norleucinal and lactacystin, prevented apo[a] degradation and increased apo[a] secretion. Transfection with an HA-tagged ubiquitin construct demonstrated the accumulation of ubiquitinated apo[a] in the presence of lactacystin. These results suggest a role for the cytoplasmic proteasome in apo[a] proteolysis. Apo[a] that accumulated intracellularly in the presence of lactacystin remained sensitive to endo-B-N-glucosaminidase H, and apo[a] degradation was reversibly inhibited by brefeldin A, suggesting that transport to a post-endoplasmic reticulum (ER) pre-medial Golgi compartment is required for apo[a] degradation. Newly synthesized apo[a] bound to the ER chaperone calnexin and conditions that enhanced this interaction prevented apo[a] degradation, suggesting that calnexin can protect apo[a] from proteolysis. These studies provide further support for the role of the proteasome in endoplasmic reticulum quality control, and expand this role to one that influences plasma levels of the atherogenic lipoprotein Lp[a].-White, A. L., B. Guerra, J. Wang, and R. E. Lanford. Presecretory degradation of apolipoprotein[a] is mediated by the proteasome pathway.

The needs of women with substance use problems: an expanded vision for treatment.

Although the number of females served in United States treatment programs for substance use has increased over the last decade, women continue to be underrepresented. This suggests that the prevalent treatment models, which tend to be male-oriented, may not provide appropriate strategies to meet women's needs. Substance use problems in women appear to be multideterminded phenomena in which genetics, familial history, psychosocial issues, and other environmental factors play contributing roles. Working from a relational theoretical model of female psychosocial development, a continuum of expanded services addressing the entire context of women's lives is discussed.

Amino terminus of apolipoprotein B suffices to produce recognition of malondialdehyde-modified low density lipoprotein by the scavenger receptor of human monocyte-macrophages.

Malondialdehyde, a product of lipid peroxidation, produces threshold conversion of low density lipoprotein (LDL) to a form recognized by type I and type II scavenger receptors of monocyte-macrophages. To investigate whether localized domains of human apoB-100 protein provide recognition determinants, we tested the ability of several different apoB-bearing particles to interact with the scavenger receptor of human monocyte-macrophages. Genetically engineered, carboxyl-terminally truncated apoB proteins assembled into lipoprotein form were labeled by fluorescent dye. Fluorescence microscopy and quantitative fluorescent spectrophotometry showed that purified particles containing as little as 23% of the apoB amino-terminus were internalized by the scavenger receptor after, but not before, malondialdehyde modification. There was no recognition of the particles by the LDL receptor. Similar results were obtained with human plasma LDL homozygous for carboxyl-terminally truncated apoB-45.2. Liposome-incorporated fusion protein containing apoB residues 547-735 displayed specific uptake by the scavenger receptor without modification by malondialdehyde. In contrast, fusion proteins containing apoB residues 3,029-3,133 or a short amino terminal segment failed to interact. Thus, primary sequence presented by residues 1-1,084 sufficed to produce recognition of modified LDL by the scavenger receptor. These receptor-combining domains were sequestered when secreted in lipoprotein form and were expressed upon malondialdehyde modification. When packaged exogenously in liposome form, fusion protein containing apoB residues 547-735, containing approximately 4% of the primary sequence, mediated scavenger receptor-dependent uptake and hydrolysis. Our findings provide an additional function or the amino-terminal region of apoB and demonstrate that primary sequence presented by the first 2% of apoB-100 protein suffices to produce recognition on malondialdehyde-modified LDL by the scavenger receptor of human monocyte-macrophages.

Influence of allelic variation on apolipoprotein(a) folding in the endoplasmic reticulum.

Plasma levels of lipoprotein(a) (Lp(a)) vary over 1000-fold between individuals and are determined by the gene for its unique apolipoprotein, apo(a), which has greater than 100 alleles. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the ability of apo(a) allelic variants to escape the endoplasmic reticulum (ER) are a major determinant of Lp(a) production rate. To examine the reason for these differences, the folding of newly synthesized apo(a) was analyzed in pulse-chase experiments. Samples were harvested in the presence of N-ethylmaleimide to preserve disulfide-bonded folding intermediates, and apo(a) was analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Apo(a) required a prolonged period (30-60 min) to reach its fully oxidized form. Multiple folding intermediates were resolved, including a disulfide-linked, apo(a)-containing complex. Unexpectedly, all allelic variants examined showed similar patterns and kinetics of folding. Even "null" apo(a) proteins, which are unable to exit the ER, appeared to fold normally. The ER glucosidase inhibitor, castanospermine, prevented apo(a) secretion, but did not inhibit folding. This suggests that an event which is dependent on trimming of N-linked glucoses, and which occurs after the folding events detectable in our assay, is required for apo(a) secretion. Differences in the ability to undergo this event may explain the variable efficiency with which apo(a) allelic variants exit the ER.

Biogenesis of Lp(a) in transgenic mouse hepatocytes.

Lipoprotein(a) [Lp(a)] biogenesis was examined in primary cultures of hepatocytes isolated from mice transgenic for both human apolipoprotein(a) [apo(a)] and human apoB. Steady-state and pulse-chase labeling experiments demonstrated that newly synthesized human apo(a) had a prolonged residence time (approximately 60 min) in the endoplasmic reticulum (ER) before maturation and secretion. Apo(a) was inefficiently secreted by the hepatocytes and a large portion of the protein was retained and degraded intracellularly. Apo(a) exhibited a prolonged and complex folding pathway in the ER, which included incorporation of apo(a) into high molecular weight, disulfide-linked aggregates. These folding characteristics could account for long ER residence time and inefficient secretion of apo(a). Mature apo(a) bound via its kringle domains to the hepatocyte cell surface before appearing in the culture medium. Apo(a) could be released from the cell surface by apoB-containing lipoproteins. These studies are consistent with a model in which the efficiency of post-translational processing of apo(a) strongly influences human plasma Lp(a) levels, and suggest that cell surface assembly may be one pathway of human Lp(a) production in vivo. Transgenic mouse hepatocytes thus provide a valuable model system with which to study factors regulating human Lp(a) biogenesis.

Kringle-containing fragments of apolipoprotein(a) circulate in human plasma and are excreted into the urine.

Apolipoprotein(a) [apo(a)] contains multiple kringle 4 repeats and circulates as part of lipoprotein(a) [Lp(a)]. Apo(a) is synthesized by the liver but its clearance mechanism is unknown. Previously, we showed that kringle 4-containing fragments of apo(a) are present in human urine. To probe their origin, human plasma was examined and a series of apo(a) immunoreactive peptides larger in size than urinary fragments was identified. The concentration of apo(a) fragments in plasma was directly related to the plasma level of Lp(a) and the 24-h urinary excretion of apo(a). Individuals with low (< 2 mg/dl) plasma levels of Lp(a) had proportionally more apo(a) circulating as fragments in their plasma. Similar apo(a) fragments were identified in baboon plasma but not in conditioned media from primary cultures of baboon hepatocytes, suggesting that the apo(a) fragments are generated from circulating apo(a) or Lp(a). When apo(a) fragments purified from human plasma were injected intravenously into mice, a species that does not produce apo(a), apo(a) fragments similar to those found in human urine were readily detected in mouse urine. Thus, we propose that apo(a) fragments in human plasma are derived from circulating apo(a)/Lp(a) and are the source of urinary apo(a).

Neomycin inhibits secretion of apolipoprotein[a] by increasing retention on the hepatocyte cell surface.

Neomycin therapy reduces plasma levels of low density lipoprotein and lipoprotein[a] (Lp[a]). To determine whether neomycin directly alters the biogenesis of Lp[a], we have examined the effect of neomycin on apolipoprotein[a] (apo[a]) synthesis and secretion in primary cultures of baboon hepatocytes. Using this system, we have previously shown that apo[a] is synthesized as a lower molecular weight precursor that upon maturation becomes associated with the cell surface before release into the culture medium. Treatment of hepatocytes with 10 mM neomycin reduced levels of apo[a] in the culture medium by as much as 12-fold. Although a portion of the reduced secretion could be accounted for by a reduction in total protein synthesis, the greatest effect of neomycin on apo[a] secretion was to decrease the release of mature apo[a] from the hepatocyte cell surface into the culture medium. Treatment of hepatocyte cultures with trypsin confirmed that mature apo[a] in neomycin-treated cells was still transported to the cell surface. Examination of related antibiotics demonstrated that inhibition of apo[a] secretion is a general property shared by the deoxystreptamine antibiotics. The mechanism by which neomycin affects the apo[a]-cell surface interaction is not known, but neomycin is known to perturb cell surface membranes, inhibit the interaction of some ligands with their cell surface receptors, and inhibit the metabolism of phosphatidylinositol 4,5 biphosphate. These studies suggest that cell surface association of apo[a] may play a role in Lp[a] biogenesis in vivo.

High prevalence of colorectal cancer in HLA-B27 transgenic F344 rats with chronic inflammatory bowel disease.

BACKGROUND/AIMS: Transgenic rats expressing the human major histocompatibility class I molecule HLA-B27 develop a spontaneous multisystem disease that includes a chronic colitis resembling ulcerative colitis. The availability of this phenotype in B27 transgenic rats of 2 different inbred strains provided the opportunity to inquire whether colorectal neoplasia, which occurs with increased frequency in humans with inflammatory bowel disease (IBD), would develop in either or both rat genetic backgrounds. METHODS: Clinical and histologic evaluation of B27 transgenic rats with chronic inflammatory bowel disease (IBD) on the F344 and LEW inbred backgrounds. RESULTS: In B27 transgenic rats on an inbred F344 background, hyperplastic lesions evolved in the setting of chronic colitis, with a high frequency of colorectal polyp formation and frequent histologic progression from adenoma to adenocarcinoma. In contrast, no neoplasia occurred in B27 transgenic rats on an inbred LEW background, despite similar colitis. CONCLUSION: A high incidence of spontaneous colorectal neoplasia occurs in a line of B27 F344 rats that shares some features of both sporadic and inflammatory bowel disease-associated human colorectal cancer. This represents a novel example of spontaneous colorectal neoplasia in rodents that is not based on germline modification of one or more already-identified cancer-related genes.