Regulated export of a secretory protein from the ER of the hepatocyte: a specific binding site retaining C-reactive protein within the ER is downregulated during the acute phase response.

The half-time for secretion of the plasma protein C-reactive protein (CRP) by the hepatocyte decreases markedly in association with its increased synthesis during the acute phase response to tissue injury (Macintyre, S., D. Samols, and I. Kushner. 1985. J. Biol. Chem. 260:4169-4173). In studies in which subcellular fractions were prepared from cells incubated under pulse-chase conditions, CRP was found to be preferentially retained within the ER of normal hepatocytes, but secreted relatively efficiently in cells prepared from rabbits undergoing the acute phase response. On the basis of the detergent-dependency of specific binding of radiolabeled CRP, as well as EM visualization of biotinylated CRP identified with peroxidase-conjugated streptavidin, CRP was found to bind to the lumenal surface of permeabilized rough microsomes, while no binding was detected in Golgi fractions. As judged by both kinetic and equilibrium binding studies, rough microsomes from control rabbits were found to have two classes of specific binding sites for CRP; a high affinity site (Kd = 1 nM, Bmax = 1 pmol CRP/mg microsomal protein) as well as a much lower affinity (Kd = 140 nM) site. In contrast, only the lower affinity class was detected in microsomes isolated from rabbits undergoing the acute phase response. On nitrocellulose blots probed with radiolabeled CRP a 60-kD protein, distinct from BiP, was detected in extracts of rough microsomes isolated from control rabbits, but not in Golgi fractions or rough microsomes from stimulated animals. These findings correlate with previous observations of changes in secretion kinetics of CRP and are consistent with the hypothesis that the intracellular sorting of CRP could be rerouted by downregulation of a specific ER binding site during the acute phase response.

Ozone depletion: ultraviolet radiation and phytoplankton biology in antarctic waters.

The springtime stratospheric ozone (O3) layer over the Antarctic is thinning by as much as 50 percent, resulting in increased midultraviolet (UVB) radiation reaching the surface of the Southern Ocean. There is concern that phytoplankton communities confined to near-surface waters of the marginal ice zone will be harmed by increased UVB irradiance penetrating the ocean surface, thereby altering the dynamics of Antarctic marine ecosystems. Results from a 6-week cruise (Icecolors) in the marginal ice zone of the Bellingshausen Sea in austral spring of 1990 indicated that as the O3 layer thinned: (i) sea surface- and depth-dependent ratios of UVB irradiance (280 to 320 nanometers) to total irradiance (280 to 700 nanometers) increased and (ii) UVB inhibition of photosynthesis increased. These and other Icecolors findings suggest that O3-dependent shifts of in-water spectral irradiances alter the balance of spectrally dependent phytoplankton processes, including photoinhibition, photoreactivation, photoprotection, and photosynthesis. A minimum 6 to 12 percent reduction in primary production associated with O3 depletion was estimated for the duration of the cruise.

In vivo studies of serum C-reactive protein turnover in rabbits.

We determined the plasma half-life of the acute phase protein C-reactive protein (CRP) both in normal rabbits and in rabbits that had received inflammatory stimuli. Rabbit CRP was purified from acute phase serum by Cx-polysaccharide affinity chromatography, radiolabeled, and rendered pyrogen-free. Six unstimulated rabbits were injected intravenously with (125)1-CRP prepared by the lactoperoxidase method and four were injected with CRP labeled by methylation using [(14)C]formaldehyde. Blood samples were obtained at 0.25 h and at intervals thereafter. Plasma half-life of CRP was calculated from the data generated during the first 12 h, by which time an average of 86% of labeled protein had disappeared from the blood stream. The mean half-life for CRP was 4.45+/-0.2 h, with no significant difference (0.40 < P < 0.45) between (125)1- and (14)C-labeled CRP. In six animals stimulated with either endotoxin or turpentine 24 h before injection of labeled CRP, a mean half-life of 5.8+/-0.6 h was found, not significantly different (0.30 < P < 0.35) from unstimulated rabbits. We equated fractional catabolic rate to fractional disappearance rate, since the rate constant for passage of CRP from vascular to extravascular compartment can be assumed to be relatively small compared to the observed fractional disappearance rate. Fractional catabolic rate was independent of serum CRP concentration; average fractional catabolic rate in all 16 animals was 14+/-0.8% h(-1) of the plasma pool. We were able to estimate rate of CRP synthesis, based on steady-state assumptions of pool sizes in those rabbits whose serum CRP levels did not change substantially during the period of study. Values as low as 6.7 mug/kg per h in the unstimulated animals and as high as 560 mug/kg per h in the stimulated animals were found.

Kinetic modeling and mathematical analysis indicate that acute phase gene expression in Hep 3B cells is regulated by both transcriptional and posttranscriptional mechanisms.

To evaluate the possible role of posttranscriptional mechanisms in the acute phase response, we determined the kinetics of transcription (by nuclear run-on assay) and mRNA accumulation of five human acute phase genes in Hep 3B cells incubated with conditioned medium from LPS-stimulated monocytes. Increase in mRNA accumulation was comparable to increase in transcription rate for fibrinogen-alpha and alpha-1 protease inhibitor, suggesting largely transcriptional regulation. In contrast, mRNA accumulation was about 10-20-fold greater than transcriptional increase for serum amyloid A, C3, and factor B, suggesting participation of posttranscriptional mechanisms. Since finding a disparity between the magnitudes of increase in mRNA and transcription does not definitively establish involvement of posttranscriptional mechanisms, we subjected our data to modeling studies and dynamic mathematical analysis to evaluate this possibility more rigorously. In modeling studies, accumulation curves resembling those observed for these three mRNAs could be generated from the nuclear run-on results only if posttranscriptional regulation was assumed. Dynamic mathematical analysis of relative transcription rates and relative mRNA abundance also strongly supported participation of posttranscriptional mechanisms. These observations suggest that posttranscriptional regulation plays a substantial role in induction of some, but not all acute phase proteins.

Physical structure of lakes constrains epidemics in Daphnia populations.

Parasites are integral parts of most ecosystems, yet attention has only recently focused on how community structure and abiotic factors impact host-parasite interactions. In lakes, both factors are influenced by habitat morphology. To investigate the role of habitat structure in mediating parasitism in the plankton, we quantified timing and prevalence of a common microparasite (Metschnikowia bicuspidata) in its host, Daphnia dentifera, in 18 lakes that vary in basin size and shape. Over three years, we found substantial spatial and temporal variation in the severity of epidemics. Although infection rates reached as high as 50% in some lakes, they did not occur in most lakes in most years. Host density, often considered to be a key determinant of disease spread, did not explain a significant amount of variation in the occurrence of epidemics. Furthermore, host resistance does not fully explain this parasite's distribution, since we easily infected hosts in the laboratory. Rather, basin shape predicted epidemics well; epidemics occurred only in lakes with steep-sided basins. In these lakes, the magnitude of epidemics varied with year. We suggest that biological (predation) and physical (turbulence) effects of basin shape interact with annual weather patterns to determine the regional distribution of this parasite.

Export of altered forms of an Escherichia coli K-12 outer membrane protein (OmpA) can inhibit synthesis of unrelated outer membrane proteins.

Expression of mutant ompA genes, encoding the 325 residue Escherichia coli outer membrane protein OmpA, caused an inhibition of synthesis of the structurally unrelated outer membrane porins OmpC and OmpF and of wild-type OmpA, but not of the periplasmic beta-lactamase. There was no accumulation of precursors of the target proteins and the inhibitory mechanism operated at the level of translation. So far only alterations around residue 45 of OmpA have been found to affect this phenomenon. Linkers were inserted between the codons for residues 45 and 46. A correlation between size and sequence of the resulting proteins and presence or absence of the inhibitory effect was not found, indicating that the added residues acted indirectly by altering the conformation of other parts of the mutant OmpA. To be effective, the altered polypeptides had to be channelled into the export pathway. Internal deletions in effector proteins, preventing incorporation into the membrane, abolished effector activity. The results suggest the existence of a periplasmic component that binds to OmpA prior to membrane assembly; impaired release of this factor from mutant OmpA proteins may trigger inhibition of translation. The factor could be a See B-type protein, keeping outer membrane proteins in a form compatible with membrane assembly.

Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12.

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12. A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed. Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized. Intact cells possessing these proteins were treated with proteases. Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion. Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action. Thus, the former area is cell surface exposed while the latter area appears not to be. The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth. It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.

Gender differences in the associations between health and neighbourhood environment.

Multiple deprivation indicators are frequently used to capture the characteristics of an area. This is a useful approach for identifying the most deprived areas, and summary indices are good predictors of mortality and morbidity, but it remains unclear which aspects of the residential environment are most salient for health. A further question is whether the most important aspects vary for different types of residents. This paper focuses on whether associations with neighbourhood characteristics are different for men and women. The sociopolitical and physical environment, amenities, and indicators of economic deprivation and affluence were measured in neighbourhoods in the UK, and their relationship with self-rated health was investigated using multilevel regression models. Each of these contextual domains was associated with self-rated health over and above individual socioeconomic characteristics. The magnitude of the association was larger for women in each case. Statistically significant interactions between gender and residential environment were found for trust, integration into wider society, left-wing political climate, physical quality of the residential environment, and unemployment rate. These findings add to the literature indicating greater effects of non-work-based stressors for women and highlight the influence of the residential environment on women's health.

Bilateral deep brachial vein thrombophlebitis due to vibrio fetus.

A patient had bilateral deep brachial vein thrombophlebitis in which Vibrio fetus was recovered from six blood cultures of the six drawn. Fever and phlebitis continued with treatment with intravenous doses of heparin and oxacillin but rapidly improved with treatment change of oxacillin to clindamycin. In vitro antibiotic disk susceptibility testing confirmed resistance to oxacillin and susceptibility to clindamycin. Vibrio fetus infection is associated with the vascular endothelium in this and previously reported cases

'What does the phrase "safer sex" mean to you?' Understanding among Glaswegian 18-year-olds in 1990.

OBJECTIVE: To ascertain what young people in western Scotland understand by the phrase 'safer sex', and the social correlates of this knowledge. DESIGN: Face-to-face interviews with 18-year-olds participating in the third stage of a longitudinal study of the social patterning of health and illness. SETTING: The Central Clydeside Conurbation in western Scotland. PARTICIPANTS: Eight hundred and seventy-nine 18-year-olds (416 men and 463 women). MAIN OUTCOME MEASURES: Reported understanding of the term 'safer sex'. RESULTS: Eighty-four per cent mentioned condoms, 68% some aspect of choice of partner, and only 2% abstaining from specific sexual activities. Those in more advantaged socio-economic circumstances and with higher levels of education were more likely to have 'better' knowledge (defined as mentioning condom use plus one or more other risk-reduction strategy). CONCLUSIONS: Although knowledge of the routes of transmission of HIV may be reasonably good among the young, knowledge of what professionals mean by 'safer sex' appears less so, particularly among the more deprived sections of the community.

Free thyroxine estimates in nonthyroidal illness: comparison of eight methods.

Eight methods for estimating free serum T4 were compared for 26 patients with nonthyroidal illness (NTI) and 16 hypothyroid patients with comparable total T4 (TT4) concentrations. Free T4 values were determined by equilibrium dialysis, enzyme immunoassay (Abbott), antibody-coated tube (Clinical Assays), antibody-coated microfine silica (Corning Immunophase), microencapsulated antibody (Damon), and free T4 index using the T3 uptake ratio or thyroxine-binding globulin method. Equilibrium dialysis, Clinical Assays and Abbott methods usually provided free T4 estimates in the normal range in NTI patients with low TT4 values and differentiated them from hypothyroid patients with comparable TT4 levels. In contrast, the other methods gave decreased free T4 estimates in the low TT4-NTI groups and often did not distinguish them from hypothyroid patients. The normal free T4 estimates by equilibrium dialysis, Clinical Assays, and Abbott methods in the low TT4-NTI patients are consistent with the previous findings of normal T4 disposal rates in these patients. These three methods may assist the clinician in differentiating the low T4 state of NTI from overt thyroxine deficiency of hypothyroidism.

Cloning and study of the genetic organization of the exe gene cluster of Aeromonas salmonicida.

The Aeromonas salmonicida (As) exe gene cluster, an additional member of the pul-related operon family required for general signal-sequence-dependent secretion of proteins from Gram- bacteria, was cloned in the broad-host-range cosmid pLAFR3. Twelve genes, exeC-N, were identified by partial nucleotide (nt) sequence analyses (exeE-N) or determination of the complete sequence (exeC and exeD). The organisation of the exeC-N genes is similar to that of several other operons of this family. These genes are arranged contiguously and are apparently transcribed in the same direction. On alignment of As and A. hydrophila exe sequences a 73-bp 'silent' deletion was identified close to the end of the As exeF gene. No gene encoding prepilin peptidase (the PulO homolog) was detected in this region. The exeN gene is evidently the last gene of this operon; it is followed by an ORF encoding a putative transcription regulator.

Topological analysis of the amino-terminal region of lactose permease using the Escherichia coli outer membrane protein, OmpA, as a marker.

LacY-ompA fusions, encoding the N-terminal 50, 71 or 143 residues of lactose permease, were constructed. The observed orientation of the OmpA part of each hybrid protein with respect to the plasma membrane supports current models of the N-terminus of Lac permease. Hybrids possessing the entire mature OmpA were very stable; those with only a part thereof were much less stable. Due to their in vivo stability and accessibility to antibody it is proposed that such hybrids may represent potential models to investigate the assembly pathway of lactose permease.

Specific high affinity binding of human interleukin 1 beta by Caf1A usher protein of Yersinia pestis.

Understanding the interaction of Yersinia pestis with the key components of the immune system is important for elucidation of the pathogenesis of bubonic plague, one of the most severe and acute bacterial diseases. Here we report the specific, high affinity binding (Kd = 1.40 x 10(-10) M +/- 0.14 x 10(-10)) of radiolabelled human interleukin 1 beta (hIL-1 beta) to E. coli cells carrying the capsular f1 operon of Y. pestis. Caf1A outer membrane usher protein was isolated to greater than 98% purity. Competition studies with purified Caf1A, together with immunoblotting studies, identified Caf1A as the hIL-1 beta receptor. Competition between Caf1 subunit and hIL-1 beta for the same or an overlapping binding site on Caf1A was demonstrated. Relevance of these results to the pathogenesis of Y. pestis and other Gram negative bacterial pathogens with homologous outer membrane usher proteins is discussed.

The role of the mature part of secretory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli.

Presently available data are reviewed which concern the role of the mature parts of secretory precursor proteins in translocation across the plasma membrane of Escherichia coli. The following conclusions can be drawn; i) signals, acting in a positive fashion and required for translocation do not appear to exist in the mature polypeptides; ii) a number of features have been identified which either affect the efficiency of translocation or cause export incompatibility. These are: alpha) protein folding prior to translocation; beta) restrictions regarding the structure of N-terminus; gamma) presence of lipophilic anchors; delta) too low a size of the precursor. Efficiency of translocation is also enhanced by binding of chaperonins (SecB, trigger factor, GroEL) to precursors. Binding sites for chaperonins appear to exist within the mature parts of the precursors but the nature of these sites has remained rather mysterious. Mutant periplasmic proteins with a block in release from the plasma membrane have been described, the mechanism of this block is not known. The mature parts of secretory proteins can also be involved in the regulation of their synthesis. It appears that exported proteins are already recognized as such before they are channelled into the export pathway and that their synthesis can be feed-back inhibited at the translational level.

Study of the intergenic exeF-exeG region and its application as a simple preliminary test for Aeromonas spp.

The exeF-exeG intergenic regions from different hybridization groups (HG) of Aeromonas were studied by PCR amplification using a single pair of primers. Six main classes of PCR products were identified according to size: 360 bp, 320 bp, 280 bp, 230-240 bp, 220 bp and 160 bp. Direct sequencing of the PCR products indicated that the shorter intergenic regions had probably originated from deletion of DNA segments between direct repeats. Correlation of certain PCR products with Aeromonas caviae (HG4), A. caviae (HG5), A. veronii (HG8) and A. salmonicida (HG3) was revealed. The PCR reaction was also shown to be generally specific for Aeromonas spp. Thus, the usefulness of this rapid, single colony-based PCR test for both identification and preliminary differentiation of Aeromonas spp. is demonstrated.

Gender differences in minor morbidity among full time employees of a British university.

STUDY OBJECTIVE: To examine gender differences in minor morbidity among men and women working in similar circumstances, and to test whether the relation between reported working conditions and health is similar for men and women. DESIGN: Multivariate analysis of data collected from a postal questionnaire distributed to full time employees in white collar jobs within a single organisation. SETTING: A British university. PARTICIPANTS: 1641 employees (1009 men and 632 women) working full time in white collar occupations in the university. MAIN RESULTS: Overall, female university employees reported more "physical" symptoms (2.0 v 1.7, p < 0.001) and more "malaise" symptoms (1.4 v 1.1, p < 0.001) than male employees, but mean scores on a measure of minor psychiatric morbidity did not differ by gender. Poor perceived working conditions (and particularly lack of job stimulation, job drain and poor physical conditions) were consistently related to all three measures of minor morbidity, and these variables accounted for most of the variance in these health measures in this sample. When the analysis controlled for occupational grade, perceived working conditions and orientation to gender roles, there was no difference between men and women for any of the health measures. The relations for the predictor variables were generally the same for men and women (and there were no interactions with gender for any of the work related variables). CONCLUSIONS: Although small gender differences in recent experience of malaise and physical symptoms remain when examining men and women in as similar working circumstances as possible, these differences are cumulatively eroded by taking account of occupational grade, reported working conditions and orientation to gender. These results lend support to a differential exposure, rather than a differential vulnerability, model of gender differences in health.

Correlates of blood pressure in 15 year olds in the west of Scotland.

STUDY OBJECTIVE: The aim was to examine social and physical correlates of blood pressure in 15 year olds. DESIGN: This was the first, baseline, sweep of a longitudinal survey of 15 year olds based on a two stage stratified clustered random sample. SETTING: The Central Clydeside Conurbation, in the West of Scotland. In 1981 this had a population of 1.7 million and a standardised mortality ratio (relative to Scotland as a whole) of 109. SUBJECTS: A random sample of households containing 15 year olds were approached by Strathclyde Regional Council; 70% agreed to have their names passed on to the MRC (15% refused, 10% could not be contacted, and 5% had moved). Of these 1177, 11% refused to participate, 3% were not contactable/had moved, and 4% did not provide full data. Complete blood pressure data are available for 959 15 year olds (464 males and 495 females). MEASUREMENTS AND MAIN RESULTS: Blood pressure, pulse rate, height, weight, and room temperature were measured by nurses in the subjects' homes. Smoking, drinking, and frequency of vigorous exercise were self reported. Maternal height, birthweight, occupation of head of household, and housing tenure were reported by parents. After controlling for the other variables, systolic blood pressure was significantly associated with weight, pulse rate, and room temperature in males and with weight, pulse rate, housing tenure, smoking, and exercise in females. Diastolic blood pressure was associated with room temperature in males and with mother's height, pulse rate, and housing tenure in females. Controlling for current weight, birthweight was inversely related to systolic blood pressure in males and positively associated in females, though in neither case were these associations statistically significant. CONCLUSIONS: In males, blood pressure was mainly related to anthropometric factors whereas in females it was additionally related to socioeconomic and behavioural variables. Although not reaching significance, the weight standardised relationship between birthweight and systolic blood pressure was consistent for males, but not females, with those reported by recent British studies of children and adults. The longitudinal design of this study will allow us to examine correlates of blood pressure in the same individuals as they reach social and physical maturity.

Do housing tenure and car access predict health because they are simply markers of income or self esteem? A Scottish study.

OBJECTIVE: To investigate relations between health (using a range of measures) and housing tenure or car access; and to test the hypothesis that observed relations between these asset based measures and health are simply because they are markers for income or self esteem. DESIGN: Analysis of data from second wave of data collection of West of Scotland Twenty-07 study, collected in 1991 by face to face interviews conducted by nurse interviewers. SETTING: The Central Clydeside Conurbation, in the West of Scotland. SUBJECTS: 785 people (354 men, 431 women) in their late 30s, and 718 people (358 men, 359 women) in their late 50s, participants in a longitudinal study. MEASURES: General Health Questionnaire scores, respiratory function, waist/hip ratio, number of longstanding illnesses, number of symptoms in the last month, and systolic blood pressure; household income adjusted for household size and composition; Rosenberg self esteem score; housing tenure and care access. RESULTS: On bivariate analysis, all the health measures were significantly associated with housing tenure, and all except waist/hip ratio with car access; all except waist/hip ratio were related to income, and all except systolic blood pressure were related to self esteem. In models controlling for age, sex, and their interaction, neither waist/hip ratio nor systolic blood pressure remained significantly associated with tenure or care access. Significant relations with all the remaining health measures persisted after further controlling for income or self esteem. CONCLUSIONS: Housing tenure and car access may not only be related to health because they are markers for income or psychological traits; they may also have some directly health promoting or damaging effects. More research is needed to establish mechanisms by which they may influence health, and to determine the policy implications of their association with health.