A peptide binding protein having a role in antigen presentation is a member of the HSP70 heat shock family.

The T cell recognition of globular protein antigens requires the processing and presentation of the antigen by Ia-expressing APCs. Processing is believed to involve the uptake of antigen into an acidic compartment where proteolysis occurs. The resulting peptides containing the T cell antigenic determinant are associated with Ia and presented at the cell surface to the specific T cells. The mechanisms by which antigenic peptides become associated with Ia is not known. We previously described a peptide binding protein of 72/74 x 10(3) Mr (PBP72/74) that plays a role in antigen presentation as shown by the ability of an antiserum raised in rabbits to affinity-purified PBP72/74 to block presentation of cytochrome c to a cytochrome c-specific T cell hybrid. Here we show that PBP72/74 is recognized by mAbs specific for members of the HSP70 family of proteins. In Western blots PBP72/74 is bound by mAb 7.10, specific for an evolutionarily conserved epitope of HSP proteins and by mAb N27, specific for both the constitutively expressed and inducible 72/73 x 10(3) Mr HSP70 proteins. In addition, PBP72/74 shares a second common feature of the HSP proteins, that of binding to ATP. Indeed, ATP causes the release of PBP72/74 from binding to a peptide fragment of cytochrome c (Pc 81-104) and PBP72/74 can be eluted from ATP columns by Pc 81-104. Finally, a portion of PBP72/74 is shown to be present on B cell surfaces by immunofluorescence staining. Thus, it appears that characteristics of the heat shock proteins are shared by a protein playing a role in antigen presentation, suggesting some commonality in function.

Ocean biogeochemistry modeled with emergent trait-based genomics.

Marine ecosystem models have advanced to incorporate metabolic pathways discovered with genomic sequencing, but direct comparisons between models and "omics" data are lacking. We developed a model that directly simulates metagenomes and metatranscriptomes for comparison with observations. Model microbes were randomly assigned genes for specialized functions, and communities of 68 species were simulated in the Atlantic Ocean. Unfit organisms were replaced, and the model self-organized to develop community genomes and transcriptomes. Emergent communities from simulations that were initialized with different cohorts of randomly generated microbes all produced realistic vertical and horizontal ocean nutrient, genome, and transcriptome gradients. Thus, the library of gene functions available to the community, rather than the distribution of functions among specific organisms, drove community assembly and biogeochemical gradients in the model ocean.

Major and minor epitopes on the self antigen mouse cytochrome c mapped by site-directed mutagenesis.

Variants of rat (mouse) cytochrome c, prepared by site-directed mutagenesis or represented by closely-related cytochromes c from different species, were employed to map the functional boundaries of a number of mouse monoclonal antibodies (mAb) specific for the major antigenic region on the self antigen (Ag) around residue 62 and the minor antigenic region around residue 44. The recombinant mouse cytochromes c tested were, unlike the tissue-derived Ag, trimethylated at position 72, and included the wild-type which was acetylated at the amino terminus, a variant that was unacetylated at the amino terminus, and variants with the following single amino acid residue replacements: V11I (valine to isoleucine at position 11), Q12M, A15S, A44P, F46Y, D50A, T58I and G89E. Of these, only the A44P variant affected the binding of mAb to the region previously localized to the vicinity of residue 44, thus confirming that assignment. Loss of the acetyl group at the amino terminus affected the binding of most of the mAb to the region around residue 62. The other mutations had little, if any, affect on mAb binding. The epitopes of mAb binding the region around residue 62 were shown in this study to have similar functional boundaries. This site on the self Ag, which encompasses at least three discontinuous segments of the polypeptide chain, is comparable in size to epitopes on other protein Ag that have been mapped by X-ray crystallography and is similar to an epitope in the corresponding region of the foreign Ag, horse cytochrome c, that has been mapped by hydrogen-deuterium exchange. In addition to the mAb binding the regions around residues 44 and 62, a third group of mouse cytochrome c-specific mAb known to be broadly reactive with mammalian cytochromes c and that represents a minor portion of the mAb was tested for binding the site-directed mutants of mouse cytochrome c. None of these mAb were affected by the mutations, indicating the presence of at least one more antigenic region on the self Ag in an area not encompassed by these mutations that is structurally highly conserved.

Stimulation of megakaryocytopoiesis in mice by human modified C-reactive protein (mCRP)

The prototypic human acute phase reactant, C-reactive protein (CRP), and a structurally modified form of CRP (mCRP) were studied as agents which could stimulate thrombopoiesis in both in vitro and in vivo mouse models. mCRP, but not the widely studied (native) pentameric form of CRP, demonstrated significant megakaryocyte colony-stimulating activity. This activity was measured in plasma clot cultures incubated with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). mCRP increased the number of mouse megakaryocyte colonies in a dose-dependent manner. While significantly more colonies were observed in mCRP-treated cultures compared to controls, the kinetics of megakaryocyte growth and maturation were similar to those measured in cultures stimulated with PWM-SCM lacking mCRP. A low level of megakaryocyte growth-promoting activity was noted when mCRP was added to plasma clot cultures not incubated with spleen cell conditioned medium. However, the most striking activity of mCRP was in potentiating stimulated megakaryocyte colony formation (i.e., as a Meg-POT factor). In in vivo experiments, mCRP injected subcutaneously into normal mice resulted in significant increases in blood platelet numbers compared to control mice receiving sham injections. These results suggest that a modified form of CRP has thrombopoietic activity in both in vitro and in vivo mouse models, Therefore, one important biological role for CRP during an acute-phase response might be to contribute, after a structural modification, to the hematopoietic regulation of blood platelets.

Measurement of conjugated diene lipids by derivative spectroscopy in heptane extracts of plasma.

A series of experiments are described which show that second derivative spectroscopy can be used to quantify conjugated lipid dienes as markers of lipid peroxidation in heptane extracts of plasma from patients with rheumatoid arthritis, osteoarthritis, and healthy controls. Results obtained by this method gave reasonable agreement with those derived from the measurement of simple absorbance in chloroform/methanol extracts. Two minima were observed in the derivative spectrum of plasma lipid extracts. These minima occurred at 233 and 241 nm and corresponded to absorbance maxima in the conventional UV spectrum. Using a combination of phospholipase hydrolysis, reverse phase high performance liquid chromatography (HPLC) and second derivative spectroscopy we confirmed that these two minima can be attributed to a single fatty acid (9 cis-, 11 trans-linoleic acid) shown previously to account for greater than 90% of diene conjugation in human plasma samples. When the biological isomer 9 cis-, 11 trans-linoleic acid was separated by reverse phase HPLC from the mixture of other plasma phospholipid-2-esterified fatty acids we observed a change in derivative spectroscopy minima from 233 and 241 nm to 228 and 237 nm. Minima at the latter two wavelengths were also seen with pure preparations of the Paint Research Isomer (9 trans-, 11 trans-linoleic acid) which eluted later than biological 9 cis-, 11 trans-linoleic acid using reverse phase HPLC, suggesting that the absorption spectra of these pure cis-, trans and trans, trans dienes are similar but can be altered by the presence of other fatty acids in the extract.

The use of different lipids to express serum tocopherol: lipid ratios for the measurement of vitamin E status.

Plasma tocopherol was measured in 85 alcoholic patients and 40 control subjects from a local factory. Cholesterol, triglycerides and phospholipids were measured individually and summed to give an estimate of total serum lipids. Plasma tocopherol concentrations of the alcoholics were significantly lower than those of the controls and showed wide variation from marked deficiency to the upper limit of the normal range. Using regression analysis, 1.11 mumol tocopherol/mmol total lipids were calculated as the threshold of deficiency equivalent to 0.8 mg tocopherol/g total lipid established by Horwitt et al. The sensitivity and specificity of other tocopherol:lipid ratios for identifying vitamin E deficiency was compared with the tocopherol:total lipid ratio. Thresholds of deficiency for the different tocopherol:lipid ratios were calculated. The tocopherol:cholesterol+triglyceride ratio was found to be almost as powerful in identifying vitamin E deficiency as the tocopherol:total lipid ratio (sensitivity 95%, specificity 99%). Of the tocopherol:individual lipid ratios, the tocopherol:cholesterol ratio gave the best results (sensitivity 86%, specificity 94%).

The influence of needle size on post-myelography headache: a controlled trial.

This study aimed to test the hypothesis that the use of fine rather than larger calibre needles for myelography reduces the incidence of post-procedural headache. 160 patients were randomized in a prospective controlled trial of 22 gauge versus 26 gauge needles. The incidence and overall severity of headache as measured at 1, 4 and 21 days after myelography was not significantly different in the two groups.

Fundholding in general practice and financial risk.

OBJECTIVE: To estimate the financial effect of random yearly variations in need for services on fundholding practices with various list sizes. DESIGN: A simulation model was derived using historical data on general practitioner referrals for the 113 surgical procedures covered by the general practitioner fund, combined with data on the hospital prices for those procedures. PATIENTS: Resident population of Central Birmingham Health Authority. MAIN OUTCOME MEASURES: Expected expenditure on the relevant surgical procedures for the whole district and for practices with list sizes of 9000, 12,000, 15,000, 18,000, 21,000, or 24,000 for each of 100 simulated years. RESULTS: By using average hospital prices for the West Midlands region the mean (SD) annual expenditure for the 179,400 residents was 4,832,471 pounds (87,149 pounds); the random variation between the 5th and 95th most expensive years was 5.7% of the mean cost. For a practice with a list size of 9000 the values were 244,891 pounds (18,349 pounds), with a variation of 27.5%. With a list size of 24,000 the values were 652,762 pounds (32,512 pounds), with a variation of 15.3%. CONCLUSIONS: Random variations in need for inpatient services will have a significant financial impact on the practice fund. The problem will be particularly great for smaller practices. Additional measures are required to ensure that the scheme is not undermined and that the potential benefits are secured.

Transferring the costs of expensive treatments from secondary to primary care.

General practitioners, especially fundholders, are becoming increasingly concerned about being asked to prescribe treatments for their patients that are outside their therapeutic experience. They are concerned about the clinical responsibility for such prescribing and the effects on their budgets. In some specialties transferring the costs of expensive treatments from secondary to primary care (cost shifting) has become partly institutionalised because of the separate sources of funding for drugs prescribed in the two sectors. With increased efforts to control the rising costs of the drugs budget and the emergence of new expensive treatments, cost shifting will be a challenge to clinicians and purchasers as they strive for rational, cost effective prescribing. A review of the funding mechanisms for drugs prescribing and of the relation between the licensing process and the decision to support the use of a treatment in primary or secondary care is needed.

Pharmacokinetics and tissue penetration of ciprofloxacin.

A 500-mg dose of the quinoline ciprofloxacin was administered orally to each of six healthy male volunteers, after which the concentrations of this agent in serum and blister fluid were measured. Absorption appeared to be rapid, with a mean peak level of 2.4 micrograms/ml attained 1.25 h after administration. The serum elimination half-life was 3.9 h. The agent penetrated blister fluid well, the percent penetration being 57%. Urinary recovery of ciprofloxacin was 30%.

How long should patients with Salmonella typhi or Salmonella paratyphi be followed-up? A comparison of published guidelines.

Records were examined for 242 individuals infected with Salmonella typhi or S. paratyphi identified in Birmingham between 1981 and 1988, with a total of 335 person years of follow-up. Of these cases 77 and 78 per cent respectively were followed beyond the point at which surveillance would have ceased under guidelines published by the American Public Health Association and by the Public Health Laboratory Service for England and Wales. Under these two sets of guidelines only seven (3.8 per cent) and eight (4.3 per cent) cases respectively had subsequent positive faecal or urine cultures over a median of 335 and 295 days of additional follow-up. After 0, 1, 2, 3, 4 and 5 prior consecutive negative sets of cultures obtained at weekly intervals the likelihood of the next set of cultures being positive was 26, 9, 5, 2.2, 2.4 and 0 per cent respectively. Only 38 (1.7 per cent) of 2184 follow-up urine cultures were positive; these results did not influence duration of follow-up. Only 26 (2.6 per cent) of 1002 contacts were infected; the yields of the first, second and third sets of cultures were 1.5, 0.6 and 0.5 per cent respectively.

The pharmacokinetics and tissue penetration of norfloxacin.

The pharmacokinetics and cantharides-induced blister fluid levels of norfloxacin were studied after a single 400 mg oral dose. The mean maximum serum level was 1.45 mg/l and occurred 1.5 h after administration. The serum half-life of norfloxacin was found to be 3.5 h. After 24 h 27% of the administered dose was recovered in the urine as microbiologically active compound. High urine levels were found. Rapid blister fluid penetration occurred, the maximum level (occurring between 2-3 h) was about 1 mg/l. Thereafter the blister fluid level exceeded the serum level, both declining in parallel.

Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia river, its estuary, and the adjacent coastal ocean.

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the alpha, gamma, or delta subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (beta-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and alpha-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.

Further evidence of lipid peroxidation in post-enteropathic haemolytic-uraemic syndrome.

Lipid peroxidation may play a role in the pathogenesis of the haemolytic-uraemic syndrome (HUS). Thirteen children with the post-enteropathic form of HUS were studied using conjugated diene lipids as markers of in vivo lipid peroxidation. Levels of total conjugated diene lipids and 9,11-linoleic acid, the principal conjugated diene in human plasma, were greater in the acute phase of this disorder than in controls. The ratio of plasma vitamin E to lipid was lower than that in children with other renal diseases, and the expected positive correlation between vitamin E and lipids did not hold for HUS patients. These data provide further evidence of lipid peroxidation in HUS and a disturbance in the metabolism of the principal lipid-bound anti-oxidant vitamin E.

Population screening for abdominal aortic aneurysm: do the benefits outweigh the costs?

To prevent the high mortality rate associated with ruptured abdominal aortic aneurysm (AAA), population screening at or soon after retirement age has been advocated, with elective operations being performed on patients with the appropriate indications. There is considerable pressure on some health authorities to fund such programmes even though there is substantial uncertainty about the consequent benefits. The ultrasound screening test is acceptable and accurate. Also, other health problems may be detected in the same screen. However, screening would lead to a questionable increase in surgery, as most patients with AAA die from other causes and not from a ruptured aneurysm. In addition, there is an elective operative mortality of around 5 per cent. Furthermore, as many of those who have a positive result on screening would never have known that they harbour an aneurysm, there is the possibility of unnecessary anxiety arising from the test. An economic analysis has been conducted for two identical, hypothetical cohorts of men using the best available data. One cohort was assumed to undergo screening and a number of men were indicated for immediate elective aneurysm repair or for follow-up, and surgery if their aneurysms become large. The other cohort was assumed not to be screened and would thereby face the possibility of rupture with its adverse outcome. The survival prospects of the two cohorts were calculated as life expectancies and in terms of life-years: the incremental life-years gained were compared with the incremental costs of the programme. Although there are considerable uncertainties in the analysis parameters, the base-line result and sensitivity analysis indicate that, on the basis of current knowledge, population screening should not be introduced.

Evidence for lipid peroxidation in man following paraquat ingestion.

Four patients were investigated for evidence of lipid peroxidation between 4.5 and 36 h (mean 22 h) after ingestion of paraquat by measuring plasma phospholipid-2-esterified, diene-conjugated 18:2 delta 9,11-linoleic acid (9,11-LA) and expressing it also as a ratio R (9,11-LA/9,12-LA X 100) of the 'parent' linoleic acid. The mean value for R was 4.73 (range 3.7-7.1) at presentation and 6.91 at peak values (range 3.8-13.4) which occurred at a mean of 34 h after ingestion. Both values were significantly higher (P less than 0.001) than that of 107 healthy controls (1.94, range 0.67-3.8). Parallel changes in plasma vitamin E and 9,11-LA occurred in the 2 patients in whom serial measurements were made suggesting an involvement of vitamin E in the formation of this isomer. These findings support the hypothesis that lipid peroxidation occurs during paraquat poisoning in man and the early appearance of 9,11-LA suggests that it may be a primary event.

Alcohol metabolism and toxicity: role of cytochrome P-450.

A new isozyme of cytochrome P-450, designated from 3a on the basis of its relative electrophoretic mobility, has been purified to homogeneity from liver microsomes of rabbits treated chronically with ethanol. This cytochrome has the highest activity of the known rabbit P-450 isozymes in the oxidation of ethanol to acetaldehyde. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozyme 3a, 4, and 6, the three major forms of cytochrome P-450 present in liver microsomes from rabbits chronically treated with ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.

Immunochemical identification of cytochrome P-450 isozyme 3a (P-450ALC) in rabbit nasal and kidney microsomes and evidence for differential induction by alcohol.

Polyclonal and monoclonal antibodies to rabbit liver microsomal alcohol-inducible cytochrome P-450 isozyme 3a (P-450ALC) were used to examine the tissue distribution of the cytochrome. Isozyme 3a or an immunochemically indistinguishable variant of this protein was detected on immunoblots of kidney and nasal mucosa microsomes, but not of microsomes prepared from brain, lung, adrenal, heart, intestine, ovary, spleen, testis, or uterus from untreated or ethanol-treated rabbits. The presence of isozyme 3a was also indicated by inhibition by anti-3a IgG of microsomal aniline hydroxylation and butanol oxidation. The identity of isozyme 3a was further substantiated by peptide-mapping analysis of the immunoaffinity-purified proteins. The amount of isozyme 3a was increased in kidney, but not in nasal microsomes, by chronic ethanol treatment. The induction of isozyme 3a in the kidney was reflected in a more than 2-fold increase in the total rates and a 7-fold increase in the isozyme 3a-dependent rates of aniline and butanol metabolism. Based on immunoblot quantitation, the specific content of isozyme 3a is about 10 pmol/mg of protein in kidney and 80 pmol/mg of protein in nasal microsomes of untreated rabbits. After ethanol treatment of the animals, the content increases to 50 pmol/mg of protein in kidney but is unchanged in nasal microsomes. The presence of isozyme 3a may play a significant role in the toxicity of foreign compounds.