Metabolic demands of a simulated smoke-diving drill.

The main goal of this study was to update the Finnish smoke-diving drill (FSDD) and to measure the physical strain of and recovery from the drill. Furthermore, the aim was to compare the physical strain of contract and professional firefighters and effect of floor materials. The associations between aerobic capacity and physical strain were also studied. The updates made included an added hose pull task and updating the equipment used. Heart rate (HR), oxygen consumption (V̇O2), and blood lactate concentration ([La-]) of 32 professional and 5 contract firefighters were measured before, during, and 10 and 30 min after the updated drill. The mean HR during the drill was 78% and V̇O2 59% of maximum. HR and [La-] had not recovered to baseline levels after 30-minute recovery period. Physical strain was higher among contract firefighters and [La-] accumulation on rough floor surfaces. Better aerobic capacity was associated with reduced physical strain.

The purpose of this study was to update the Finnish smoke-diving drill. This paper describes the process of updating the drill, and the experimental measurements regarding the metabolic demands of the updated drill. The updates made included adding a hose pull task and updating the equipment used during the drill.

Interventions to enhance recovery in healthy workers; a scoping review.

BACKGROUND: Faster recovery from work may help to prevent work-related ill health. AIMS: To provide a preliminary assessment of the range and nature of interventions that aim to improve recovery from cognitive and physical work. METHODS: A scoping review to examine the range and nature of the evidence, to identify gaps in the evidence base and to provide input for systematic reviews. We searched for workplace intervention studies that aimed at enhancing recovery. We used an iterative method common in qualitative research to obtain an overview of study elements, including intervention content, design, theory, measurements, effects and cost-effectiveness. RESULTS: We found 28 studies evaluating seven types of interventions mostly using a randomized controlled study design. For person-directed interventions, we found relaxation techniques, training of recovery experiences, promotion of physical activity and stress management. For work-directed interventions, there were participatory changes, work-break schedules and task variation. Most interventions were based on the conservation of resources and affect-regulation theories, none were based on the effort-recovery theory. The need for recovery (NfR) and the recovery experiences questionnaires (REQ) were used most often. Study authors reported a beneficial effect of the intervention in 14 of 26 published studies. None of the studies that used the NfR scale found a beneficial effect, whereas studies that used the REQ showed beneficial effects. Three studies indicated that interventions were not cost-effective. CONCLUSIONS: Feasible and possibly effective interventions are available for improving recovery from cognitive and physical workload. Systematic reviews are needed to determine their effectiveness.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

This corrects the article DOI: 10.1038/cdd.2016.22.

Transferrin-Targeted Nanoparticles Containing Zoledronic Acid as a Potential Tool to Inhibit Glioblastoma Growth.

The treatment of glioblastoma (GBM) is a challenge for the biomedical research since cures remain elusive. Its current therapy, consisted on surgery, radiotherapy, and concomitant chemotherapy with temozolomide (TMZ), is often uneffective. Here, we proposed the use of zoledronic acid (ZOL) as a potential agent for the treatment of GBM. Our group previously developed self-assembling nanoparticles, also named PLCaPZ NPs, to use ZOL in the treatment of prostate cancer. Here, we updated the previously developed nanoparticles (NPs) by designing transferrin (Tf)-targeted self-assembling NPs, also named Tf-PLCaPZ NPs, to use ZOL in the treatment of brain tumors, e.g., GBM. The efficacy of Tf-PLCaPZ NPs was evaluated in different GBM cell lines and in an animal model of GBM, in comparison with PLCaPZ NPs and free ZOL. Tf-PLCaPZ NPs were characterized by a narrow size distribution and a high incorporation efficiency of ZOL. Moreover, the presence of Tf significantly reduced the hemolytic activity of the formulation. In vitro, in LN229 cells, a significant uptake and cell growth inhibition after treatment with Tf-PLCaPZ NPs was achieved. Moreover, the sequential therapy of TMZ and Tf-PLCaPZ NPs lead to a superior therapeutic activity compared to their single administration. The results obtained in mice xenografted with U373MG, revealed a significant anticancer activity of Tf-PLCaPZ NPs, while the tumors remained unaffected with free TMZ. These promising results introduce a novel type of easy-to-obtain NPs for the delivery of ZOL in the treatment of GBM tumors.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Association of cardio-ankle vascular index with physical fitness and cognitive symptoms in aging Finnish firefighters.

PURPOSE: Monitoring cardiovascular risk factors is important in health promotion among firefighters. The assessment of arterial stiffness (AS) may help to detect early signs of atherosclerosis. The aim of this study was to analyze associations between aerobic fitness, cognitive symptoms and cardio-ankle vascular index (CAVI) as a measure for AS among Finnish firefighters. METHODS: The data are one part of a large 13-year follow-up study of the health and physical and mental capacity of Finnish professional firefighters. The subjects in this substudy comprised 65 male firefighters of a mean age of 48.0 (42-58) years in 2009. Their maximal oxygen uptake was successfully measured in two cross-sectional studies in 1996 and 2009, and they responded to questionnaires at both sessions, and their CAVI was measured in 2009. CAVI was calculated from the pulse waveform signal and pulse wave velocity. The lifestyle habits and subjective cognitive stress-related symptoms were collected via a standardized questionnaire. Muscular fitness was measured by the routine test battery used for Finnish firefighters. RESULTS: CAVI was related to age. About one-fifth of the firefighters had a CAVI of >8. Aerobic fitness was the main physiological factor correlating with increased CAVI. Interestingly, VO(2)max and the accelerated decrease in VO(2)max during a 13-year follow-up were associated with signs of impaired vascular function. The cognitive symptoms derived from the Profile of Mood States questionnaire (POMS) were mainly associated with stress and sleeping difficulties. No clear association with physical fitness was found in this population of fit firefighters. CONCLUSIONS: Among firefighters, the decrease in aerobic fitness predicts increased arterial stiffness. The speed of the age-related decline in maximal oxygen consumption is as important as absolute level. Against expectations, the cognitive function did not correlate with vascular health parameters. The cognitive symptoms, however, were only mild.

Caveolins and membrane cholesterol.

Caveolae (small plasma membrane invaginations) and their coat proteins, caveolins, have attracted the attention of researchers in diverse fields, including cell biology, cardiovascular and cancer research. The tight association between caveolin and cholesterol governs the biochemical behaviour of caveolae and is emerging as an important characteristic in a number of processes assigned to these multifunctional organelles. In this review, selected aspects of the caveolin-cholesterol association and its potential functional implications are discussed.

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface.

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15 degrees C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

Direct observation of lipoprotein cholesterol ester degradation in lysosomes.

We have investigated whether pyrene-labelled cholesterol esters (PyrnCEs) (n indicates the number of aliphatic carbons in the pyrene-chain) can be used to observe the degradation of low-density lipoprotein (LDL)-derived cholesterol esters (CEs) in the lysosomes of living cells. To select the optimal substrates, hydrolysis of the PyrnCE species by lysosomal acid lipase (LAL) in detergent/phospholipid micelles was compared. The rate of hydrolysis varied markedly depending on the length of the pyrenyl chain. Pyr10CE was clearly the best substrate, while Pyr4CE was practically unhydrolysed. Pyr10CE and [3H]cholesteryl linoleate, the major CE species in LDL, were hydrolysed equally by LAL when incorporated together into reconstituted LDL (rLDL) particles, thus indicating that Pyr10CE is a reliable reporter of the lysosomal degradation of native CEs. When rLDL particles containing Pyr4CE or Pyr10CE were incubated with fibroblasts, the accumulation of bright intracellular vesicular fluorescence was observed with the former fluorescent derivative, but not with the latter. However, when the cells were treated with chloroquine, an inhibitor of lysosomal hydrolysis, or when cells with defective LAL were employed, Pyr10CE also accumulated in vesicular structures. HPLC analysis of cellular lipid extracts fully supported these imaging results. It is concluded that PyrnCEs can be used to observe degradation of CEs directly in living cells. This should be particularly useful when exploring the mechanisms responsible for the accumulation of lipoprotein-derived CEs in complex systems such as the arterial intima.

Degradation of low density lipoprotein cholesterol esters by lysosomal lipase in vitro. Effect of core physical state and basis of species selectivity.

The effect of the physical state of low density lipoprotein (LDL) core and the selectivity of the degradation of LDL cholesterol esters (CEs) by the lysosomal acid lipase (LAL) in vitro were investigated. The physical state of LDL was modulated by varying temperature or the triglyceride content of the core. Normal LDL showed an abrupt increase of CE hydrolysis at 24 degrees C and another deviation occurred close to 36 degrees C. 1H-NMR measurements showed that these temperatures coincide with the onset and end temperatures of the LDL core lipid transition, respectively. Enrichment of LDL with triglycerides abolished the abrupt changes both in the CE hydrolysis and in the physical state of LDL lipids. These findings show that there is a correlation between the physical state of LDL lipids and the rate of LAL-mediated hydrolysis of the CEs in the particle. The relative rates of hydrolysis of different CE species were also compared. With native LDL, increasing the length of a saturated acyl chain from 14 to 20 carbons reduced the rate of degradation of CE modestly, while increasing acyl chain unsaturation increased the rate of degradation markedly. However, cholesterol oleate was hydrolyzed more slowly than cholesterol stearate. Essentially the same order of hydrolytic susceptibility was observed when the CE species were incorporated into triglyceride-enriched LDL, reconstituted high density lipoprotein particles or in detergent/phospholipid micelles. These results indicate that the selective hydrolysis of CE species in LDL is determined mainly by the ease with which the CE molecule can emerge from the surface layer reach the active site of LAL. Slower degradation of the more saturated CEs by LAL could lead, under certain conditions, to their accumulation in lysosomes and eventually, to cell death, lysis and deposition of crystalline, poorly mobilizable lipids to the arterial intima.

Human arterial proteoglycans increase the rate of proteolytic fusion of low density lipoprotein particles.

Low density lipoprotein (LDL) particles can undergo fusion in the arterial intima, where they are bound to proteoglycans. Here we studied the effect of human arterial proteoglycans on proteolytic fusion of LDL in vitro. For this purpose, an assay was devised based on fluorescence resonance energy transfer that allowed continuous monitoring of fusion of proteoglycan-bound LDL particles. We found that addition of human arterial proteoglycans markedly increased the rate of proteolytic fusion of LDL. The glycosaminoglycans isolated from the proteoglycans also increased the rate of fusion, demonstrating that this effect was produced by the negatively charged sulfated polysaccharides in the proteoglycans. Furthermore, heparin, chondroitin 6-sulfate, and dextran sulfate, three commercially available sulfated polysaccharides, also increased the rate of LDL fusion, with heparin and chondroitin 6-sulfate being as effective as and dextran sulfate more effective than human proteoglycans. The ability of the sulfated polysaccharides to increase the rate of proteolytic fusion of LDL depended critically on their ability to form insoluble complexes with LDL, which, in turn, resulted in an increased rate of LDL proteolysis and, in consequence, in an increased rate of LDL fusion. The results reveal a novel mechanism regulating LDL fusion and point to the potentially important role of arterial proteoglycans in the generation of LDL-derived lipid droplets in the arterial intima during atherogenesis.

Degradation of pyrene-labelled phospholipids by lysosomal phospholipases in vitro. Dependence of degradation on the length and position of the labelled and unlabelled acyl chains.

The hydrolysis of pyrenylacyl phosphatidylcholines (PyrnPCs)(n indicates the number of aliphatic carbons in the pyrene-chain) by crude lysosomal phospholipases in vitro was investigated. PyrnPCs consist of several sets in which the length of the pyrene-labelled or the unlabelled acyl chain, linked to the sn-1 or sn-2 position, was systematically varied. Lysophosphatidylcholine and fatty acid were the only fluorescent breakdown products detected, thus indicating that PyrnPCs were degraded by A-type phospholipases and lysophospholipases. Of these, mainly A1-type phospholipases appear to be involved, as determined from the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and position of the pyrene-labelled and unlabelled chains it is suggested that (1) the lysosomal A-type phospholipases acting on PyrnPCs recognize the carboxy-terminal part of the lipid acyl chains and (2) the relevant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are peroxidized and polymerized, may not be good substrates for the lysosomal phospholipases mentioned. The impaired hydrolysis of the most hydrophobic PyrnPCs indicates that lysosomal phospholipases may not be able to penetrate significantly into the substrate interphase, but upward movement of the lipid may be required for efficient hydrolysis. Finally, the rate of hydrolysis of many pyrenyl derivatives was found to be comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivatives can be used as faithful reporters of lysosomal degradation of natural lipids in vivo and in vitro.

The mechanism of human plasma phospholipid transfer protein-induced enlargement of high-density lipoprotein particles: evidence for particle fusion.

1. Phospholipid transfer protein (PLTP) mediates conversion of high-density lipoprotein (HDL3) to large particles, with concomitant release of apolipoprotein A-I (apoA-I). To study the mechanisms involved in this conversion, reconstituted HDL (rHDL) particles containing either fluorescent pyrenylacyl cholesterol ester (PyrCE) in their core (PyrCE-rHDL) or pyrenylacyl phosphatidylcholine (PysPC) in their surface lipid layer (PyrPC-rHDL) were prepared. Upon incubation with PLTP they behaved as native HDL3, in that their size increased considerably. 2. When PyrPC-rHDL was incubated with HDL3 in the presence of PLTP, a rapid decline of the pyrene excimer/monomer fluorescence ratio (E/M) occurred, demonstrating that PLTP induced mixing of the surface lipids of PyrPC-rHDL and HDL3. As this mixing was almost complete before any significant increase in HDL particle size was observed, it represents PLTP-mediated phospholipid transfer or exchange that is not directly coupled to the formation of large HDL particles. 3. When core-labelled PyrCE-rHDL was incubated in the presence of PLTP, a much slower, time-dependent decrease of E/M was observed, demonstrating that PLTP also promotes mixing of the core lipids. The rate and extent of mixing of core lipids correlated with the amount of PLTP added and with the increase in particle size. The enlarged particles formed could be visualized as discrete, non-aggregated particles by electron microscopy. Concomitantly with the appearance of enlarged particles, lipid-poor apoA-I molecules were released. These data, together with the fact that PLTP has been shown not to mediate transfer of cholesterol esters, strongly suggest that particle fusion rather than (net) lipid transfer or particle aggregation is responsible for the enlargement of HDL particles observed upon incubation with PLTP.4.ApoA-I rHDL, but not apoA-II rHDL, were converted into large particles, suggesting that the presence of apoA-I is required for PLTP-mediated HDL fusion. A model for PLTP-mediated enlargement of HDL particles is presented.

Are the job demands on physical work capacity equal for young and aging firefighters?

The job demands on physical work capacity and the frequency of the firefighting and rescue tasks were rated by 156 professional firefighters (age range, 22 to 54 years) who responded to a questionnaire. Smoke-diving requiring the use of personal protective equipment was considered to demand most aerobic power. The clearing of debris with heavy manual tools, and roof work set the highest demands on muscular performance and motor coordination, respectively. During the past 5 years, 83 to 88% of the respondents had performed these tasks on average four times a year. The rating and frequency of the tasks were not significantly affected by age. The results suggest that the job demands on physical work capacity remain the same throughout the occupational career of the firefighters.

Physiological responses of firefighting students during simulated smoke-diving in the heat.

While wearing a self-contained breathing apparatus and fire-protective clothing, 35 healthy firefighting students aged 19-27 years performed smoke-diving (entry into a smoke-filled room) during a simulated shipboard fire. The mean (+/- SD) ambient temperature inside the simulator was 119 +/- 12 degrees C, and the task lasted 17 +/- 4 min. All subjects were fit according to their maximal oxygen consumption, which was 52.4 +/- 5.2 mL/min/kg (4.08 +/- 0.45 l/min). During the smoke-diving the average heart rate was 150 +/- 13 beats/min (79 +/- 6% of maximal heart rate attained in a cycle-ergometer test), and the peak heart rate was 180 +/- 13 beats/min (95 +/- 6% of maximal heart rate). The estimated oxygen consumption was 2.4 +/- 0.5 L/min (60 +/- 12% of maximal oxygen consumption). Neither ability to tolerate stress (as determined by the instructors) nor previous experience in smoke-diving tasks seemed to influence the heart rate or estimated oxygen consumption during experiment. Smoke-diving was physically very demanding even for the young and fit subjects, showing the importance of regular evaluation of the health and physical fitness of every firefighter who has to carry out smoke-diving tasks.

The effect of appraisers in estimating metabolic rate with the Edholm scale.

A study was made of the appraisers' effect on the estimation of metabolic rate with the Edholm scale and a table of the ISO 7243 heat stress standard. The appraisers, five experienced and five inexperienced persons, estimated the metabolic rate of three different work tasks from videotapes. Analysis of variance indicated significant ( [Formula: see text] ) differences in the appraisers' recordings of the activities. The appraisers were grouped according to the similarity of the estimated values they gave. The groups thus contained both experienced and inexperienced appraisers, and it was not possible to classify the appraisers into experienced and inexperienced groups according to their earlier experience. The metabolic rates according to the Edholm scale were higher than according to the ISO 7243 table. The differences in metabolic rates given by the individual observers varied from 38 to 118 W/m(2). The variations in the estimation of metabolic rates were greater when the Edholm scale was used. This variation caused considerable variation also in the predicted mean vote, PMV index. It is recommended that the appraisers be selected carefully, because it is not possible to know whether a randomly selected appraiser is an 'average' or an 'extreme' appraiser without a test. Before conducting extensive field surveys where several appraisers estimate the metabolic rates, it would be useful to arrange training in order to calibrate the levels of the Edholm scale as well as ISO method among the appraisers because training clearly unified the estimation.

Elongation of both branches of biantennary backbones of oligo-(N-acetyllactosamino)glycans by human serum (1----3)-N-acetyl-beta-D- glucosaminyltransferase.

Partial reactions catalyzed by a (1----3)-N-acetyl-beta-D- glucosaminyltransferase (EC2.4.1.149), known to be present in human serum, were studied by use of biantennary "backbone" saccharides of oligo-N-acetyllactosamine-type as acceptors. Incubation of the radiolabeled blood-group I-active hexasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp- (1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-GlcNAc (1) and UDP-GlcNAc with serum gave first a transient 1:1 mixture of two isomeric heptasaccharides, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D- Galp-(1----4)-D-GlcNAc (2) and beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----3)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-Glc NAc (3), showing that both branches of 1 react equally well. The two heptasaccharides reacted further in the incubation mixture to form the radiolabeled octasaccharide, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[be ta-D- GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Ga lp- (1----4)-D-GlcNAc (4); during this second reaction, the composition of the heptasaccharide mixture remained unchanged, indicating that 2 and 3 reacted at approximately equal rates. The heptasaccharides 2 and 3 could not be separated from each other, but they could be detected, identified, and quantitatively determined by stepwise enzymic degradations. Partial (1----3)-N-acetyl-beta-D-glucosaminylation reactions, carried out with another acceptor, the branched pentasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Gal (11), revealed that it reacted also equally well at both branches.(ABSTRACT TRUNCATED AT 400 WORDS)

Fate of the 6K membrane protein of Semliki Forest virus during virus assembly.

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.