Polycystic ovary syndrome and risk of stillbirth: a nationwide register-based study.

OBJECTIVE: To investigate whether polycystic ovary syndrome (PCOS) is associated with increased risk of stillbirth and whether any such association is linked to PCOS with a severe hyperandrogenic profile. DESIGN: Nationwide register-based cohort study. SETTING: Sweden. POPULATION: The cohort consisted of women giving birth to singleton infants in 1997-2015. All women with a diagnosis of PCOS in the period 1997-2017 and a randomly selected reference group of women without PCOS diagnosis were included. PCOS with a severe hyperandrogenic profile was defined as a PCOS diagnosis with at least two dispensations of prescribed anti-androgens during 2005-2017. METHODS: The risk of stillbirth in women with PCOS was estimated through multiple logistic regression, using women without PCOS as a reference. Risks were expressed as adjusted odds ratios (aORs) with 95% confidence intervals (95% CIs), adjusted for maternal age, parity, body mass index, type-1 diabetes, educational level and country of birth. MAIN OUTCOME MEASURES: Stillbirth, at ≥22 weeks of gestation in 2008-2015 and at ≥28 weeks of gestation in 1997-2007. RESULTS: Compared with women without PCOS (n = 241 750), women with PCOS (n = 41 851) had a 50% increased risk of stillbirth (aOR 1.50, 95% CI 1.28-1.77). The incidence of stillbirth in women with PCOS was particularly increased at term. Women with PCOS and a severe hyperandrogenic profile (n = 13 713) did not have a stronger association with stillbirth than women with PCOS who did not have such a profile. CONCLUSIONS: PCOS is associated with stillbirth and should be considered as a possible risk factor in antenatal care. Further research is warranted to investigate possible causal mechanisms. TWEETABLE ABSTRACT: Women with PCOS have increased risk of stillbirth, and the incidence is particularly increased at term.

Non-fasting bioelectrical impedance analysis in cystic fibrosis: Implications for clinical practice and research.

BACKGROUND: Nutritional status affects pulmonary function in cystic fibrosis (CF) patients and can be monitored by using bioelectrical impedance analysis (BIA). BIA measurements are commonly performed in the fasting state, which is burdensome for patients. We investigated whether fasting is necessary for clinical practice and research. METHODS: Fat free mass (FFM) and fat mass (FM) were determined in adult CF patients (n = 84) by whole body single frequency BIA (Bodystat 500) in a fasting and non-fasting state. Fasting and non-fasting BIA outcomes were compared with Bland-Altman plots. Pulmonary function was expressed as Forced Expiratory Volume at 1 s percentage predicted (FEV1%pred). Comparability of the associations between fasting and non-fasting body composition measurements with FEV1%pred was assessed by multiple linear regression. RESULTS: Fasting FFM, its index (FFMI), and phase angle were significantly lower than non-fasting estimates (-0.23 kg, p = 0.006, -0.07 kg/m2, p = 0.002, -0.10°, p = 0.000, respectively). Fasting FM and its index (FMI) were significantly higher than non-fasting estimates (0.22 kg, p = 0.008) 0.32%, p = 0.005, and 0.07 kg/m2, (p = 0.005). Differences between fasting and non-fasting FFM and FM were <1 kg in 86% of the patients. FFMI percentile estimates remained similar in 83% of the patients when measured after nutritional intake. Fasting and non-fasting FFMI showed similar associations with FEV1%pred (ß: 4.3%, 95% CL: 0.98, 7.70 and ß: 4.6%, 95% CI: 1.22, 8.00, respectively). CONCLUSION: Differences between fasting and non-fasting FFM and FM were not clinically relevant, and associations with pulmonary function remained similar. Therefore, BIA measurements can be performed in a non-fasting state.

Prenatal exposures and birth indices, and subsequent risk of polycystic ovary syndrome: a national registry-based cohort study.

OBJECTIVE: To study the associations between prenatal exposures and risk of developing polycystic ovary syndrome (PCOS). DESIGN: National registry-based cohort study. SETTING: Sweden. POPULATION: Girls born in Sweden during the years 1982-1995 (n = 681 123). METHODS: The girls were followed until the year 2010 for a diagnosis of PCOS. We estimated the associations between maternal body mass index (BMI), smoking, and size at birth with the risk of developing a PCOS diagnosis. Risks were calculated by adjusted hazard ratio (aHR) and 95% confidence intervals (95% CIs). MAIN OUTCOME MEASURES: A diagnosis of PCOS at 15 years of age or later. RESULTS: During the follow-up period 3738 girls were diagnosed with PCOS (0.54%). Girls with mothers who were overweight or obese had 1.5-2.0 times higher risk of PCOS (aHR 1.52, 95% CI 1.36-1.70; aHR 1.97, 95% CI 1.61-2.41, respectively), compared with girls born to mothers of normal weight. The risk of PCOS was increased if the mother smoked during pregnancy (1-9 cigarettes/day, aHR 1.31, 95% CI 1.18-1.47; ≥10 cigarettes/day, aHR 1.44, 95% CI 1.27-1.64). Being born small for gestational age (SGA) was associated with a later diagnosis of PCOS in crude estimates, but the association was not significant after adjusting for maternal factors. CONCLUSIONS: Maternal smoking and increased BMI appear to increase the risk of PCOS in offspring. The association between SGA and the development of PCOS appears to be mediated by maternal factors. TWEETABLE ABSTRACT: Smoking during pregnancy and high maternal BMI are associated with PCOS diagnosis in the offspring.

The effects of a multispecies probiotic on migraine and markers of intestinal permeability-results of a randomized placebo-controlled study.

BACKGROUND/OBJECTIVES: Migraine, associated with several gastrointestinal disorders, may result from increased intestinal permeability, allowing endotoxins to enter the bloodstream. We tested whether probiotics could reduce migraine through an effect on intestinal permeability and inflammation. SUBJECTS/METHODS: In total, 63 patients were randomly allocated to the probiotic (n=31) or the placebo group (n=32). Participants ingested a multispecies probiotic (5x109 colony-forming units) or placebo daily for 12 weeks. Migraine was assessed with the Migraine Disability Assessment Scale (MIDAS), the Headache Disability Inventory (HDI) and headache diaries. At baseline and 12 weeks, intestinal permeability was measured with the urinary lactulose/mannitol test and fecal and serum zonulin; inflammation was measured from interleukin (IL) -6, IL-10, tumor necrosis factor-α and C-reactive protein in serum. RESULTS: The MIDAS migraine intensity score significantly decreased in both groups (P<0.001) and the HDI score significantly decreased in the probiotic group (P=0.032) and borderline in the placebo group (P=0.053). In the probiotics group, patients had a median of 6 migraine days in the first month, 4 in the second month (P=0.002) and 5 in the last month, which was not significantly different from the 5, 4, and 4 days in the placebo group. A ⩾2day reduction in migraine days was seen in 12/31 patients in the probiotics group versus 7/29 in the placebo group (ns). Probiotic use did not significantly affect medication use, intestinal permeability or inflammation compared to placebo. CONCLUSIONS: In this study, we could not confirm significant benefit from a multispecies probiotic compared to a placebo on the outcome parameters of migraine and intestinal integrity.

Dietary counselling and nutritional support in oropharyngeal cancer patients treated with radiotherapy: persistent weight loss during 1-year follow-ups.

BACKGROUND/OBJECTIVES: The need for dietary counselling and nutritional support in oropharyngeal cancer patients is generally accepted. However, evidence for the effectiveness is sparse. The aim of this study was to describe dietary counselling, nutritional support, body weight and toxicity during and after treatment, and investigate the effect of pre-treatment body mass index (BMI) on survival in oropharyngeal cancer patients. SUBJECTS/METHODS: A retrospective chart review was made in 276 oropharyngeal cancer patients treated with radiotherapy (RT). End points were dietary consultations, weight loss, toxicity, overall survival and disease-free survival. RESULTS: Almost all oropharyngeal cancer patients received dietary counselling (94%) and nutritional support (99%). Dietary counselling decreased sharply shortly after treatment to 38% at 1 year after treatment. Overall weight loss increased during the first year of follow-up and ranged from 3% at start of RT, until 11% at 1 year after RT. Overall survival was significantly longer for patients with a BMI above average (P=0.01). Acute dysphagia (P=0.001), mucositis (P=0.000) and toxicity grade 3 (P=0.002) were significantly more prevalent in patients who had lost 10% or more of their body weight. CONCLUSIONS: This study showed that patients continue to lose body weight during and until 1 year after treatment, despite nutrition support and frequent dietetic consultation. A BMI above average appears to increase survival time. Future studies, preferably randomized trials, are needed to compare standard dietary counselling with more intensive dietary counselling that consists of earlier and/or prolonged treatment.

Pregnancy and perinatal outcomes in women with polycystic ovary syndrome and twin births: a population-based cohort study.

OBJECTIVE: To investigate pregnancy and perinatal outcomes in twin births among women with and without polycystic ovary syndrome (PCOS) diagnosis. DESIGN: Population-based cohort study. SETTING: Sweden. POPULATION: We identified 20,965 women with twin births between 1995 and 2009 of whom 226 had a PCOS diagnosis through linkage between the Swedish Medical Birth Register and the Swedish National Patient Register. METHODS: Calculating risk ratios (RR) with 95% confidence intervals (CI) using a log-binomial regression model and hazard ratios (HR) with 95% CI for preterm birth. MAIN OUTCOME MEASURES: Preterm birth, low birthweight, caesarean section, pre-eclampsia, Apgar score <7 at 5 minutes and perinatal mortality. RESULTS: PCOS diagnosis in twin pregnancy was associated with increased risk of preterm delivery (51% versus 43%, RR 1.18 [95% CI 1.03-1.37]), particularly spontaneous preterm delivery (37% versus 28%; RR 1.30 [95% CI 1.09-1.55]) and very preterm birth (<32 weeks) (14% versus 8%, RR 1.62 [95% CI 1.10-2.37]). Twins of PCOS mothers had more often low birthweight (48% versus 39%, adjusted RR 1.40 [95% CI 1.09-1.80]). This difference disappeared when adjusting for gestational age. No risk difference was found for caesarean section, pre-eclampsia, low 5-minute Apgar score or perinatal mortality. CONCLUSIONS: The risk of preterm delivery in twin pregnancies is increased by having a PCOS diagnosis. This should be considered in risk estimation and antenatal follow-up of twin pregnancies.

Interrelationship between milk constituents, serum oestradiol and vaginal mucus indicators of oestrus in Egyptian buffaloes.

The intensity of heat signs in buffaloes is generally low and the incidence of suboestrus varied from 15 to 73% (Buffalopedia). The objective of this study was to investigate the feasibility of monitoring the changes in some milk constituents, oestradiol levels and electrical conductivity of vaginal mucus during peri-oestrous period in prediction of the timing of oestrus in buffaloes. Twenty-one Egyptian buffaloes aged 3-9 year, 1st-6th lactations, were examined by oestrous detector and ultrasonographically for monitoring the ovarian and uterine activity for 7 days around the time of standing oestrus. Sodium, potassium, chloride and lactose were assayed in aqueous phase of milk; besides, oestradiol was estimated in serum. Current results declared highly significant acute changes in milk constituents at the time of oestrus characterized by peaking of chloride and sodium levels and lowering of potassium and lactose values. The alternation in milk composition when arranged in decreasing order of magnitude, sodium was the highest (77.78 ± 0.69%), followed by chloride (61.60 ± 1.52%) and potassium (-58.14 ± 10.89%). Concomitantly, milk lactose decreased by 26.07 ± 7.97% compared to baseline levels. Synchronously, vaginal electrical resistance (VER) showed a significant (p < 0.01) decrease, but serum oestradiol 17ß levels surged (59.93 ± 7.29 pg/ml) on day of oestrus. Serum oestradiol level was negatively correlated with VER (r = -0.577), potassium (r = -0.661), positively correlated with chloride (r = 0.707) and sodium (r = 0.579) and not correlated with lactose levels. These results for the first time suggested that the changes in constituents of milk during peri-oestrous period may be used as a practical non-invasive indicator for oestrous detection and prediction of ovulation in Egyptian buffaloes.

Effects of nutritional status and dietetic interventions on survival in Cystic Fibrosis patients before and after lung transplantation.

BACKGROUND: This study retrospectively investigated nutritional status, dietetic intervention and intake in Cystic Fibrosis (CF) patients before and after lung transplantation (LTX). METHODS: Body Mass Index (BMI), Fat Free Mass Index (FFMI) and nutritional intake were retrieved from 75 out-patients aged 15-53 years. Patients were seen every 3-4 months during the waiting list time (range 0-81 months) and up to 116 months after LTX. Survival was measured in months. RESULTS: The median BMI at baseline was 19.2 kg/m(2) (range: 15.3 to 28.4 kg/m(2)) with 29 patients (39%) below ≤18.5 kg/m(2). FFMI (measured in 65 patients) had a median of 15.2 kg/m(2) (range: 11.1 to 22.4 kg/m(2)) with 39 patients (60%) ≤16.7 kg/m(2) (men) or ≤14.6 kg/m(2) (women). Median energy intake was 2800 kcal, 239 kcal higher than the estimated energy requirement. However, 8 patients consumed ≥500 kcal less than recommended. Protein intake was 104 (range 60-187) g or 1.9 g/kg per day. Despite dietetic intervention with oral nutritional supplements (ONS) (36 patients), tube feeding (12 patients), or both (13 patients), BMI and FFMI hardly improved pre-LTX. LTX was performed in 51 patients (68%); 10 patients died during follow-up, median survival time was 41 months. A BMI ≤18.5 kg/m(2) was more prevalent in patients who died before LTX (6/9) or who died after LTX (4/10) than in patients who were still alive on the waiting list (5/15) or who survived LTX (14/41). Results for FFMI were comparable. From 6-12 months post-LTX, BMI and FFMI markedly improved, especially in underweight patients. CONCLUSION: A BMI ≤18.5 kg/m(2) and an FFMI ≤16.7 kg/m(2) (men) or ≤14.6 kg/m(2) (women) appears to impair survival in LTX candidates with CF. Patients maintained a low body weight before LTX. After LTX weight gain is achieved.

[Day-case holmium laser enucleation and mechanical morcellation of the prostate]. / Ambulante Holmiumlaserenukleation und mechanische Morcellierung der Prostata.

BACKGROUND: Holmium laser enucleation of the prostate (HoLEP) is increasingly being used as an alternative to transurethral resection of the prostate (TURP) and transurethral electrovaporisation of the prostate (TVP), the"gold standard" procedures for treating symptomatic benign prostatic hyperplasia. Mechanical tissue morcellation was introduced in 1998, and its efficiency could be improved. We investigated the efficiency and complication rate of a morcellator in a day-case setting. METHODS: From 2004 to 2008, 137 men were treated with an 80W holmium:YAG laser and subsequent tissue morcellation. RESULTS: The average patient age was 68.0+/-7.1 (51-86) years. Morcellation time was 8.6+/-6.8 (1-35) min. The weight of the resected tissue was 36.6+/-30.0 (2-175) g. The total time for the procedure was 69.6+/-28.7 (29-150) min, and the efficiency of morcellation was 5.0+/-2.6 (0.2-11.5) g/min. The complication rate due to morcellation was 4.3%. CONCLUSION: HoLEP with mechanical morcellation is safe even in a day-case setting. The total procedure time approximates that for TURP, so a longer theatre time loses significance as an argument against the method. Complications are rare and not severe. Further technical improvement is desirable.

A high menaquinone intake reduces the incidence of coronary heart disease.

BACKGROUND AND AIM: Vitamin K dependent proteins have been demonstrated to inhibit vascular calcification. Data on the effect of vitamin K intake on coronary heart disease (CHD) risk, however, are scarce. To examine the relationship between dietary vitamins K(1) and K(2) intake, and its subtypes, and the incidence of CHD. METHODS AND RESULTS: We used data from the Prospect-EPIC cohort consisting of 16,057 women, enrolled between 1993 and 1997 and aged 49-70 years, who were free of cardiovascular diseases at baseline. Intake of vitamin K and other nutrients was estimated with a food frequency questionnaire. Multivariate Cox proportional hazards models were used to analyse the data. After a mean+/-SD follow-up of 8.1+/-1.6 years, we identified 480 incident cases of CHD. Mean vitamin K(1) intake was 211.7+/-100.3 microg/d and vitamin K(2) intake was 29.1+/-12.8 microg/d. After adjustment for traditional risk factors and dietary factors, we observed an inverse association between vitamin K(2) and risk of CHD with a Hazard Ratio (HR) of 0.91 [95% CI 0.85-1.00] per 10 microg/d vitamin K(2) intake. This association was mainly due to vitamin K(2) subtypes MK-7, MK-8 and MK-9. Vitamin K(1) intake was not significantly related to CHD. CONCLUSIONS: A high intake of menoquinones, especially MK-7, MK-8 and MK-9, could protect against CHD. However, more research is necessary to define optimal intake levels of vitamin K intake for the prevention of CHD.

Skin involvement in scleroderma--where histological and clinical scores meet.

OBJECTIVES: A clinico-pathological study in diffuse systemic sclerosis (SSc) patients was performed to analyse whether the skin histological organization and the pro-fibrotic signals elicited by TGF-beta in fibroblasts vary according to the modified Rodnan skin score (mRSS). METHODS: Twenty-seven SSc patients underwent 45 skin biopsies with simultaneous measure of mRSS before or after treatment by immunosuppressive drugs, with or without autologous peripheral haematopoietic stem cell transplantation (HSCT). RESULTS: Double-blind optic microscopy analysis of the biopsies standard extracellular matrix stains allowed to define three histological subgroups: 6 with grade 1 weak fibrosis, 30 with grade 2 moderate fibrosis and 9 with grade 3 severe fibrosis. A significant (P < 0.0001) was identified between the grades of fibrosis and the mRSS. In skin fibroblast cultures, Smad3 phosphorylation levels, as well as mRNA steady-state levels of two transforming growth factor (TGF)-beta/Smad3 targets, COL1A2 and PAI-1, increased in parallel with the mRSS. When compared with pre-transplant values the degree of fibrosis observed after HSCT in the papillary and in the reticular dermis decreased in parallel with the fall in mRSS (n = 5 consecutive patients with repeated biopsies). CONCLUSIONS: The histological extent of skin fibrosis correlates closely with the mRSS. Both parameters appeared to regress after HSCT. The extent of TGF-beta signalling activation in SSc skin fibroblasts appears to parallel the severity of disease.

Coexistence of non-Hodgkin's lymphoma and non-small cell lung carcinoma: diagnosis and treatment.

Abstract. In this communication, we will present a very rare case of the coexistence of non-Hodgkin's lymphoma (NHL; low malignant lymphocytic lymphoma of the B-cell type) and a non-small-cell lung carcinoma (NSCLC). A patient with a 15-year history of NHL developed a generalized relapse of the lymphoma with an additional tumor mass in the left lower lobe of the lung. Bronchoscopy showed the evidence of the NHL. Due to non-responding chemotherapy on the lung tumor, the coexistence of a second malignancy was histologically proved in a second bronchoscopy. Resection of the lung tumor with complex lobectomy and lymphadenectomy was performed. After that, chemotherapy with four cycles of carboplatin supplemented with taxol was induced. The patient was discharged from the hospital with a stable remission of both tumor diseases. Restaging after six months showed no evidence of a tumor relapse. This is a very rare case of the coexistence of NHL and NSCLC; we will discuss the difficulty of diagnostic and treatment of both tumor diseases.

Structure and assembly of intracellular mature vaccinia virus: thin-section analyses.

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.

Nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces in Arctic animals.

In normal rat and human, most of the nuclei of hepatic parenchymal cells are centrally located in the cytoplasm. However, it is reported that the nuclei of hepatic parenchymal cells are situated at a deviated position on sinusoidal surfaces under pathological situations such as chronic hepatitis, hepatocellular carcinoma, adenomatous hyperplasia, or regeneration. During a study on the mechanism of extreme vitamin A-accumulation in hepatic stellate cells of arctic animals including polar bears, arctic foxes, bearded seals, and glaucous gulls, we noticed that these arctic animals displayed the nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces. In this study, we assessed the frequency of hepatic parenchymal cells showing the nuclear deviation on the sinusoidal surfaces in arctic animals. A significantly higher frequency of the nuclear deviation in hepatic parenchymal cells was seen in polar bears (89.8+/-3.4%), arctic foxes (68.6+/-10.5%), bearded seals (63.6+/-8.4%), and glaucous gulls (24.2+/-5.8%), as compared to that of control rat liver (9.8+/-3.5%). However, no pathological abnormality such as fibrosis or necrosis was observed in hepatic parenchymal and nonparenchymal cells of arctic animals, and there were no differences in the intralobular distribution of parenchymal cells displaying the nuclear deviation in the livers from either arctic animals and control rats. The hepatic sinusoidal littoral cells such as stellate cells or extracellular matrix components in the perisinusoidal spaces may influence the nuclear positioning and hence the polarity and intrinsic physiological function of parenchymal cells.

How many surgeons does a province need, and how do we determine appropriate numbers?

The study compared each province's supply of surgeons in three specialities (ophthalmologists--orthopedic--surgeons, and cardiac and thoracic surgeons) with the rates of key procedures (cataract removal, hip and knee replacement, and coronary artery bypass) that residents received. We found little or no relationship between the supply of surgeons and a population's surgery rate. We conclude that the supply of surgical specialists is the wrong focus for health care resource planning.

Characteristics of patients with a regular source of care.

This study was designed to describe patient characteristics associated with having a regular source of care among all patients who received care from large urban clinics in Manitoba over a three-year period (N = 298,222). Using administrative data, patients were classified as having a regular source of care if they made 75% or more of their total ambulatory visits to the same clinic. Overall, 44.2% of patients had a regular source of care. A logistic regression showed that children and adults aged 45 and older were more likely to have a regular source of care than patients aged 18-44. Moreover, patients with a regular source of care tended to live in more affluent neighbourhoods and were healthier than individuals with no regular source of care. Systemic changes might be needed to enhance continuity of care (e.g., mechanisms to enhance access) among vulnerable segments of the population like the poor.

Minimal exclusion of plasma membrane proteins during retroviral envelope formation.

The retrovirus forms its envelope by budding at the plasma membrane (PM). This process is primarily driven by its cytoplasmic core-precursor protein, Gag, as shown by the efficient formation of virus-like Gag particles in the absence of its envelope protein, Env. Most interestingly, several studies have demonstrated incorporation of various PM proteins into retrovirus, but the underlying mechanism of this phenomenon has remained elusive. We have purified Moloney murine leukemia virus Gag particles by sedimentation in an iodixanol gradient and donor PMs by flotation in a sucrose gradient and compared their protein compositions at equal lipid basis. We found that most PM proteins are present at similar density in both membranes. The inclusion of PM proteins was unaffected by incorporation of Env protein into the envelope of the Gag particles and whether these were produced at high or low level in the cells. These findings indicate that most PM proteins become incorporated into the retrovirus envelope without significant sorting. This feature of retrovirus assembly should be considered when studying retrovirus functions and developing retrovirus vectors.

Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure.

The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.