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.

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.

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).

Intracellular processing of apo(a) in primary baboon hepatocytes.

We have developed a serum-free medium for the long-term culture of highly differentiated primary baboon hepatocytes. Hepatocytes isolated from animals with defined plasma Lp(a) levels and apo(a) glycoprotein phenotypes were used to study the assembly of Lp(a). A combination of steady-state and pulse-chase labeling studies and endoglycosidase digests demonstrated that apo(a) was synthesized as a lower molecular weight precursor. After a prolonged period of time in the endoplasmic reticulum, apo(a) was converted to a mature form and secreted. A proportion of mature apo(a) also had a prolonged residence time in the trans Golgi apparatus. In all experiments, apoB co-immunoprecipitated with apo(a) from the culture medium but not from the cell lysates, supporting an extracellular association of the proteins for the formation of Lp(a). Analysis of hepatic RNA from 29 'null' Lp(a) phenotype baboons revealed that one-third of the animals had detectable apo(a) transcripts, whereas the remainder had no detectable apo(a) mRNA. The baboon hepatocyte system therefore represents a valuable model to examine the effect of allelic variation at the apo(a) locus on Lp(a) assembly.

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.

A comparison of hypothalamic cell numbers in dwarf and normal weight rats exposed prenatally to methylazoxymethanol (MAM).

Growth deficiencies follow MAM exposure during the period when the growth hormone releasing factor (GRF) cells of the hypothalamus form, while animals exposed slightly later in gestation when the inhibitors of growth hormone release are forming, exhibit giantism. Counts of sample regions of the hypothalamus have shown that rats treated in utero on the 14th day of gestation have reductions in the number of GRF cells, increases in the number of SRIF (somatotropin release inhibiting factor) cells, and alterations of pituitary structure. These effects occurred in all treated subjects, even though obvious effects on body size were present in a small fraction of the treated animals. The present study was designed to examine the effect of 20 mg/kg MAM on the 13th day of gestation (a peak period for production of GRF cells) on GRF and SRIF cell numbers, in a large sample of dwarf-treated rats, normal weight-treated rats, and controls. The results of total counts of hypothalamic cells identified by immunocytochemistry demonstrated significant reductions in GRF cells in both dwarf and normal weight rats exposed to MAM, compared to controls, with no difference between the two treated groups. Like pituitary weights, the neuron counts were significantly correlated with body weight only in dwarf animals. SRIF cell numbers were equivalent to those in controls, suggesting that the increase reported earlier may have been due to a rebound effect in proliferation rather than some response of SRIF cells to GRF cell reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of rat brain weight and pituitary weight to postnatal growth after prenatal exposure to methylazoxymethanol.

Teratogens can affect body weight in various ways, but the association of brain damage with postnatal growth abnormalities suggests a role for neuroendocrine growth-controlling systems. Growth deficiencies follow methylazoxymethanol (MAM) exposure during the period when the growth hormone releasing factor (GRF) cells of the hypothalamus form, and the pattern of growth of the animals is like that of animals deficient in growth hormone. The present studies were designed to examine the growth, body proportions, brain weight, and pituitary weight of animals treated with 20 mg/kg MAM on the 13th day of gestation, a peak period for production of GRF neurons. Among the offspring, this treatment produced about 25% dwarfs (animals smaller than the smallest control of the same sex). Significantly more females than males were categorized as dwarfs. The weight effect occurred long after birth, as is characteristic of animals and humans with growth hormone deficiency. Analyses of weights over the course of development indicated that prenatal factors, rather than factors operating between birth and weaning, predicted the adult body weight of dwarfs, while both sets of factors were significant in other animals. The growth reduction was symmetrical, as would be expected if the animals were growth hormone deficient, with an 18% reduction in weight reflecting a 6% reduction in bone length. The remaining treated animals were similar to controls in absolute weight, body proportions, and rate of growth. Neither pituitary weight nor brain weight appears to play the key role in determining which animals will exhibit growth deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathological lesions associated with Anoplocephala perfoliata at the ileo-caecal junction of horses.

The intestinal tracts of 20 horses, killed at a local abattoir and of unknown age, sex and previous clinical history were examined for the presence of Anoplocephala perfoliata attached to the ileo-caecal junction. Four horses had no tape-worms, nine had one to 20 tapeworms attached to the mucosa and seven had more than 100 attached to the mucosa. The histological changes of thickening, ulceration and eosinophil infiltration of the mucosa at the ileo-caecal junction were more severe when more than 100 parasites were present.

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.

Cell surface assembly of lipoprotein(a) in primary cultures of baboon hepatocytes.

Lipoprotein(a) (Lp(a)) consists of a low density lipoprotein particle in which apolipoprotein(a) (apo(a)), is disulfide linked to apoB. Lp(a) is produced by the liver, and high plasma levels represent an independent risk factor for cardiovascular diseases. However, pathways of production and metabolism of Lp(a) are poorly understood. We used primary cultures of baboon hepatocytes to analyze the steps involved in Lp(a) biogenesis. The results demonstrated that Lp(a) assembly was extracellular, since it was inhibited when anti-apo(a) antiserum was present in the culture medium. In addition, free apo(a) produced by hepatocytes could associate extracellularly with apoB in either very low density or low density lipoproteins. Lp(a) assembly required lysine-binding pockets in apo(a) kringles, as it was inhibited by the lysine analog, 6-amino hexanoic acid. A portion of apo(a) was also bound to the cell surface via its kringle domains and could be released into the medium by 6-amino hexanoic acid or proline. In add-back experiments, apo(a), but not Lp(a), bound to the cell surface. Addition of low density lipoprotein or very low density lipoprotein to hepatocyte cultures released apo(a) from the cell surface into the lipoprotein fraction of culture medium. We conclude that assembly of Lp(a) can occur at the cell surface. This represents one potential mechanism of Lp(a) production in vivo.

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.

Biosynthesis and metabolism of lipoprotein (a).

Significant advances have been made over the past year toward understanding the pathways of lipoprotein (a) biosynthesis and metabolism. Transcriptional and post-translational mechanisms have been identified as important determinants of plasma lipoprotein (a) levels. Assembly of lipoprotein (a) has been shown to be an extracellular event that occurs on the hepatocyte cell surface. The development of lipoprotein (a) transgenic mice has provided a valuable model to study the metabolism of lipoprotein (a).

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).

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.

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.

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.

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.

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.

Intracellular maturation of apolipoprotein[a] and assembly of lipoprotein[a] in primary baboon hepatocytes.

The glycoprotein apolipoprotein[a] (apo[a]) is present in plasma at highly variable concentrations and appears as a number of genetically determined size isoforms (400-800 kDa), disulfide linked to apoB-100 in low density lipoprotein to produce lipoprotein [a](Lp[a]). Apo[a] is synthesized by the liver, but the site of association of apo[a] and apoB and factors that regulate its production are unknown. To examine the morphogenesis of the Lp[a] particle, baboon hepatocytes expressing a single, low molecular weight isoform of apo[a] were labeled with [35S]cysteine and methionine, and apo[a] was analyzed by immunoprecipitation and SDS-PAGE. Steady-state labeling revealed two molecular weight forms of apo[a] inside the cell. Only the large form was recovered from the culture medium. Pulse-chase studies and endoglycosidase treatment revealed that the lower molecular weight form of apo[a] represented a precursor with a prolonged residence time in the endoplasmic reticulum or an early Golgi compartment, after which it was processed to the mature form. A proportion of the mature form of apo[a] was rapidly secreted after synthesis, whereas the remainder had a prolonged residence time in a late Golgi compartment. In all experiments, apoB co-precipitated with apo[a] from the culture medium, but not from cell lysates. Density gradient ultracentrifugation and immunoblot analysis revealed that the majority of apo[a] was secreted into the medium in a free form, suggesting that the association between apo[a] and apoB occurred after secretion. Regulation of the movement of apo[a] between intracellular compartments may be one mechanism by which the plasma levels of Lp[a] are influenced.