To study the overall structure of the peptidoglycan fabric of the sacculi of gram-negative and gram-positive walls, actively growing cultures of Escherichia coli and Bacillus subtilis were treated with boiling sodium dodecyl sulfate solutions. The sacculi were then treated with enzymes to eliminate proteins and nucleic acids. These intact saccoli were probed with fluorescein-labeled dextrans with a range of known molecular weights. The penetration of the probes could be monitored by the negative-staining appearance in the fluorescence microscope. At several chosen times, the molecular weight fraction that allowed barely observable entry of the fluorescein-labeled probe and the molecular weight fraction that penetrated to achieve almost, but not quite, the concentration of probe in the solution external to the sacculi were determined. From three pairs of times and molecular weights that met one or the other of these two criteria, the effective pore size could be calculated. The minimum size of protein molecule that could diffuse through the pores was also calculated. Two mathematical models, which gave essentially the same results, were used to interpret the experimental data: one for the permeation of random coils through a surface containing holes and the other for rigid spheres diffusing through water-filled cylindrical pores. The mean estimate of the effective hole radius in walls from E. coli is 2.06 nm, and that of the effective hole size in walls from B. subtilis is 2.12 nm. These results are supported by experiments in which the loss of preloaded cells was monitored. Various fluorescein-labeled dextran samples were mixed with samples of intact cell walls, held for a long time, and then diluted. The efflux of the dextrans was monitored. Neither large nor small dextrans stained under these conditions. Only with dextran samples of a sufficiently small size were the sacculi filled during the preincubation period, and only with the largest of these could the probe not escape quickly. From the pore (or mesh) size, it can be concluded that the wall fabric of both organisms has few imperfections and that the major passageway is through the smallest possible pore, or "tessera," formed by the maximal cross-linking of the peptides from glycan chain to glycan chain compatible with the degree of rotational flexibility of the chains of repeating disaccharides of N-acetyl muramic acid and N-acetyl glucosamine. A tessera is composed of two chains of eight saccharides cross-linked by two octapeptides. The size of a globular hydrophilic molecule, if it did not bind to wall components, that could pass freely through the meshwork of an unstretched sacculus of either organism is roughly 25 kDa. We stress that this is only a rough estimate, and it may be possible for proteins of less than 50 kDa to pass through the native wall during normal growth conditions.

The development of a membrane-bound structure separating DNA from other cellular components was the epochal evolutionary event that gave rise to eukaryotes, possibly occurring up to 2 billion years ago. Yet, this view of the nuclear envelope as a physical barrier greatly underestimates its fundamental impact on cellular organization and complexity, much of which is only beginning to be understood. Indeed, alterations of nuclear envelope structure and protein composition are essential to many aspects of metazoan development and cellular differentiation. Mutations in genes encoding nuclear envelope proteins cause a fascinating array of diseases referred to as "nuclear envelopathies" or "laminopathies" that affect different tissues and organ systems. We review recent work on the nuclear envelope, including insights derived from the study of nuclear envelopathies. These studies are uncovering new functions for nuclear envelope proteins and underlie an emerging view of the nuclear envelope as a critical signaling node in development and disease.

BACKGROUND

Processed copies of genes (retrogenes) are duplicate genes that originated through the reverse-transcription of a host transcript and insertion in the genome. This type of gene duplication, as any other, could be a source of new genes and functions. Using whole genome sequence data for 12 Drosophila species, we dated the origin of 94 retroposition events that gave rise to candidate functional genes in D. melanogaster.

RESULTS

Based on this analysis, we infer that functional retrogenes have emerged at a fairly constant rate of 0.5 genes per million years per lineage over the last approximately 63 million years of Drosophila evolution. The number of functional retrogenes and the rate at which they are recruited in the D. melanogaster lineage are of the same order of magnitude as those estimated in the human lineage, despite the higher deletion bias in the Drosophila genome. However, unlike primates, the rate of retroposition in Drosophila seems to be fairly constant and no burst of retroposition can be inferred from our analyses. In addition, our data also support an important role for retrogenes as a source of lineage-specific male functions, in agreement with previous hypotheses. Finally, we identified three cases of functional retrogenes in D. melanogaster that have been independently retroposed and recruited in parallel as new genes in other Drosophila lineages.

CONCLUSIONS

Together, these results indicate that retroposition is a persistent mechanism and a recurrent pathway for the emergence of new genes in Drosophila.

Ninety-three patients with malignant disease underwent orthotopic liver transplantation between May 1968 and April 1987 in the Cambridge/King's College Hospital program. Of 50 patients with primary hepatocellular carcinoma (HCC) (19 with cirrhosis, 31 without cirrhosis, including 7 with fibrolamellar variant), 37 (74%) survived for more than 3 months, and in this group evidence of tumor recurrence was obtained in 24 (64.9%), the longest survivor being 11.8 years post-transplant, and three survived for more than 5 years. Although there is no correlation between the frequency of tumor recurrence and underlying cirrhosis, or histologic type (except fibrolamellar variant), it was observed earlier in those with moderate/poorly differentiated tumors and also when prednisolone and azathioprine was used for immunosuppression. Tumor recurred in all but two of those with peripheral or central cholangiocarcinoma (one alive at 6.1 years) with median survival times of 34 weeks and 56 weeks for the central and peripheral types, respectively. Among the unusual primary tumors, one with epithelioid haemangioendothelioma developed tumor recurrence at 2 years, one of two patients with apudoma is tumor-free at 2.2 years, and the one patient with bile-duct papillary cystadenocarcinoma is alive at 1.7 years. For the secondary hepatic malignancy group, survival times were shorter with little palliation except for two patients with carcinoid syndrome who were free of associated symptoms at 6 and 10 months. Despite the overall high frequency of tumor recurrence in most categories of hepatic malignancy, liver transplantation gave worthwhile survival with a number of patients cured and in the others considerable palliation of symptoms.

BACKGROUND

Anaplastic thyroid carcinoma (ATC) is among the most aggressive of human malignancies. However, there have been few large studies of histologically well-defined ATC. We report the results of a 50-year experience of this lethal malignancy.

METHODS

We reviewed all cases of ATC managed in this institution between 1949 and 1999. One pathologist (J.R.G.) reviewed all pathologic material. Clinical details were obtained from medical records, and current status of all patients was determined.

RESULTS

There were 134 cases, with a female-to-male ratio of 1.5:1 and a mean age of 67 years. Benign thyroid disease was present in 27 cases (20%) and well-differentiated thyroid carcinoma in 31 (23%). Sixty-two patients (46%) had distant metastases at diagnosis, and 98% of the tumors were locally invasive. Primary treatment was surgical for 96 patients (72%). Complete resection was achieved in 29 cases (30%), with "minimal residual disease" in 25. Neither extent of operation nor completeness of resection affected survival (P>> .4). Postoperative radiotherapy gave slightly longer median survival (5 vs 3 months), which was not significant (P < .08). Multimodal therapy, including operation, chemotherapy, and radiotherapy, did not improve survival.

CONCLUSIONS

The outlook for patients with ATC remains grim. Novel treatments for ATC are desperately needed.

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrPSc) used limited proteolysis to digest the precursor cellular PrP (PrPC). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrPSc in all 10-24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, approximately 90% of the total PrPSc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrPC. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

A retrospective cohort of 823 patients with ulcerative colitis who resided at the time of diagnosis in one of three defined geographical areas (West Midlands region, Oxford region, England and Stockholm County, Sweden) was assembled. The patients were first seen at named hospitals in these areas and the diagnosis of ulcerative colitis established within five years of onset of symptoms between 1945-1965. All patients were 15 years of age or more at onset of disease and were followed for a minimum of 17 years and a maximum of 38 years. Ninety seven per cent completeness of follow up was achieved. Examining the colorectal cancer risk in the series relative to the risk in the general population by standardised morbidity ratios, there was an eight fold increased risk of cancer in the series as a whole. Dividing the series by extent of colitis, extensive colitis patients showed a 19 fold increase in risk. A four fold increased risk was shown in the remainder of the series (left sided colitis, proctitis and extent unknown). Life table analyses in extensive colitis gave cumulative risks of 7.2% (CI 3.6-10.8) at 20 years from onset of disease and 16.5% (CI 9.0-24.0) at 30 years from onset. No significant effect of age at onset, sex or referral centre could be detected. Examination of the data by interval from onset to cancer and by actual age at development of cancer suggests that patients who develop colorectal cancer will do so in a distribution around 50 years of age independent of duration of disease in adult onset ulcerative colitis (greater than 15 years at onset of disease). An inverse relationship was shown between age at onset of disease and interval from onset of disease to cancer. Further age specific rates for cancer increased up to 50 years and decreased thereafter. These results suggest that extensive colitis patients have a genetic predisposition to colorectal cancer and that longstanding inflammation is not of primary importance in the initiation/promotion of cancer in this disease.

SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.

OBJECTIVE

The period prevalence of depression among women is 21.9% during the first postpartum year; however, questions remain about the value of screening for depression.

OBJECTIVE

To screen for depression in postpartum women and evaluate positive screen findings to determine the timing of episode onset, rate and intensity of self-harm ideation, and primary and secondary DSM-IV disorders to inform treatment and policy decisions.

METHODS

Sequential case series of women who recently gave birth.

METHODS

Urban academic women's hospital.

METHODS

During the maternity hospitalization, women were offered screening at 4 to 6 weeks post partum by telephone. Screen-positive women were invited to undergo psychiatric evaluations in their homes.

METHODS

A positive screen finding was an Edinburgh Postnatal Depression Scale (EPDS) score of 10 or higher. Self-harm ideation was assessed on EPDS item 10: "The thought of harming myself has occurred to me" (yes, quite often; sometimes; hardly ever; never). Screen-positive women underwent evaluation with the Structured Clinical Interview for DSM-IV for Axis I primary and secondary diagnoses.

RESULTS

Ten thousand mothers underwent screening, with positive findings in 1396 (14.0%); of these, 826 (59.2%) completed the home visits and 147 (10.5%) completed a telephone diagnostic interview. Screen-positive women were more likely to be younger, African American, publicly insured, single, and less well educated. More episodes began post partum (40.1%), followed by during pregnancy (33.4%) and before pregnancy (26.5%). In this population, 19.3% had self-harm ideation. All mothers with the highest intensity of self-harm ideation were identified with the EPDS score of 10 or higher. The most common primary diagnoses were unipolar depressive disorders (68.5%), and almost two-thirds had comorbid anxiety disorders. A striking 22.6% had bipolar disorders.

CONCLUSIONS

The most common diagnosis in screen-positive women was major depressive disorder with comorbid generalized anxiety disorder. Strategies to differentiate women with bipolar from unipolar disorders are needed.

BACKGROUND

clinicaltrials.gov Identifier: NCT00282776.

OBJECTIVE

To compare lesion detection and image quality of chest computed tomographic (CT) images acquired at various tube current-time products (40-150 mAs) and reconstructed with adaptive statistical iterative reconstruction (ASIR) or filtered back projection (FBP).

METHODS

In this Institutional Review Board-approved HIPAA-compliant study, CT data from 23 patients (mean age, 63 years ± 7.3 [standard deviation]; 10 men, 13 women) were acquired at varying tube current-time products (40, 75, 110, and 150 mAs) on a 64-row multidetector CT scanner with 10-cm scan length. All patients gave informed consent. Data sets were reconstructed at 30%, 50%, and 70% ASIR-FBP blending. Two thoracic radiologists assessed image noise, visibility of small structures, lesion conspicuity, and diagnostic confidence. Objective noise and CT number were measured in the thoracic aorta. CT dose index volume, dose-length product, weight, and transverse diameter were recorded. Data were analyzed by using analysis of variance and the Wilcoxon signed rank test.

RESULTS

FBP had unacceptable noise at 40 and 75 mAs in 17 and five patients, respectively, whereas ASIR had acceptable noise at 40-150 mAs. Objective noise with 30%, 50%, and 70% ASIR blending (11.8 ± 3.8, 9.6 ± 3.1, and 7.5 ± 2.6, respectively) was lower than that with FBP (15.8 ± 4.8) (P < .0001). No lesions were missed on FBP or ASIR images. Lesion conspicuity was graded as well seen on both FBP and ASIR images (P < .05). Mild pixilated blotchy texture was noticed with 70% blended ASIR images.

CONCLUSIONS

Acceptable image quality can be obtained for chest CT images acquired at 40 mAs by using ASIR without any substantial artifacts affecting diagnostic confidence.

BACKGROUND

http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11101450/-/DC1.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor. PAF receptor is linked to intracellular signal transduction pathways, including turnover of phosphatidylinositol, elevation in intracellular calcium concentration, and activation of kinases, resulting in versatile bioactions. On the basis of numerous pharmacological reports, PAF is thought to have many pathophysiological and physiological functions. Recently advanced molecular technics enable us not only to clone PAF receptor cDNAs and genes, but also generate PAF receptor mutant animals, i.e., PAF receptor-overexpressing mouse and PAF receptor-deficient mouse. These mutant mice gave us a novel and specific approach for identifying the pathophysiological and physiological functions of PAF. This review also describes the phenotypes of these mutant mice and discusses them by referring to previously reported pharmacological and genetical data.

BACKGROUND

The HIV integrase strand transfer inhibitor elvitegravir (EVG) has been co-formulated with the CYP3A4 inhibitor cobicistat (COBI), emtricitabine (FTC), and tenofovir disoproxil fumarate (TDF) into a once-daily, single tablet. We compared EVG/COBI/FTC/TDF with a ritonavir-boosted (RTV) protease inhibitor regimen of atazanavir (ATV)/RTV+FTC/TDF as initial therapy for HIV-1 infection.

METHODS

This phase 3, non-inferiority study enrolled treatment-naive patients with an HIV-1 RNA concentration of 5000 copies per mL or more and susceptibility to atazanavir, emtricitabine, and tenofovir. Patients were randomly assigned (1:1) to receive EVG/COBI/FTC/TDF or ATV/RTV+FTC/TDF plus matching placebos, administered once daily. Randomisation was by a computer-generated random sequence, accessed via an interactive telephone and web response system. Patients, and investigators and study staff who gave treatments, assessed outcomes, or analysed data were masked to the assignment. The primary endpoint was HIV RNA concentration of 50 copies per mL or less after 48 weeks (according to the US FDA snapshot algorithm), with a 12% non-inferiority margin. This trial is registered with ClinicalTrials.gov, number NCT01106586.

RESULTS

1017 patients were screened, 715 were enrolled, and 708 were treated (353 with EVG/COBI/FTC/TDF and 355 with ATV/RTV+FTC/TDF). EVG/COBI/FTC/TDF was non-inferior to ATV/RTV+FTC/TDF for the primary outcome (316 patients [89·5%] vs 308 patients [86·8%], adjusted difference 3·0%, 95% CI -1·9% to 7·8%). Both regimens had favourable safety and tolerability; 13 (3·7%) versus 18 (5·1%) patients discontinued treatment because of adverse events. Fewer patients receiving EVG/COBI/FTC/TDF had abnormal results in liver function tests than did those receiving ATV/RTV+FTC/TDF and had smaller median increases in fasting triglyceride concentration (90 μmol/L vs 260 μmol/L, p=0·006). Small median increases in serum creatinine concentration with accompanying decreases in estimated glomerular filtration rate occurred in both study groups by week 2; they generally stabilised by week 8 and did not change up to week 48 (median change 11 μmol/L vs 7 μmol/L).

CONCLUSIONS

If regulatory approval is given, EVG/COBI/FTC/TDF would be the first integrase-inhibitor-based regimen given once daily and the only one formulated as a single tablet for initial HIV treatment.

BACKGROUND

Gilead Sciences.

OBJECTIVE

Resolvin D1 (RvD1) limits neutrophil recruitment during acute inflammation and is derived from omega-3 docosahexaenoic acid to promote catabasis. The contribution of its specific receptors, the lipoxin A(4)/Annexin-A1 receptor formyl-peptide receptor 2 (FPR2/ALX) and the orphan receptor G-protein-coupled receptor 32 (GPR32) are of considerable interest.

RESULTS

RvD1 reduced human polymorphonuclear leukocytes recruitment to endothelial cells under shear conditions as quantified using a flow chamber system. Receptor-specific antibodies blocked these anti-inflammatory actions of RvD1, with low (1 nmol/L) concentrations sensitive to GPR32 blockade, while the higher (10 nmol/L) concentration appeared FPR2/ALX-specific. Interestingly, polymorphonuclear leukocytes surface expression of FPR2/ALX but not GPR32 increased following activation with pro-inflammatory stimuli, corresponding with secretory vesicle mobilization. Lipid mediator metabololipidomics carried out with 24-hour exudates revealed that RvD1 in vivo gave a significant reduction in the levels of a number of pro-inflammatory mediators including prostaglandins and leukotriene B(4). These actions of RvD1 were abolished in fpr2 null mice.

CONCLUSIONS

Pro-resolving lipid mediators and their receptors, such as RvD1 and the 2 G-protein-coupled receptors, studied here regulate resolution and may provide new therapeutic strategies for diseases with a vascular inflammatory component.

1. Intracellular pH (pHi) was measured using pH-sensitive glass micro-electrodes. The effects on pHi of CO2 applied externally and HCO3-, H+ and NH4+ injected iontophoretically, were investigated. 2. The transport numbers for iontophoretic injection into aqueous micro-droples were found by potentiometric titration to be 0-3 for HCO3- and 0-94 for H+. 3. Exposure to Ringer, pH 7-5, equilibrated with 2-2% CO2 caused a rapid, but only transient, fall in pHi. Within 1 or 2 min pHi began to return exponentially to normal, with a time constant of about 5 min. 4. When external CO2 was removed, pHi rapidly increased, and then slowly returned to normal. The pHi changes with CO2 application or removal gave a calculated intracellular buffer value of about 30 m-equiv H+/pH unit per litre. 5. Injection of HCO3- caused a rise in pHi very similar to that seen on removal of external CO2. 6. The pHi responses to CO2 application, CO2 removal and HCO3- injection were slowed by the carbonic anhydrase inhibitor acetazolamide. 7. H+ injection caused a transient fall in pHi. In CO2 Ringer pHi fell less and recovered faster than in CO2-free Ringer. Calculation of the internal buffer value from the pHi responses to H+ and HCO3- injection gave very similar values. 8. The internal buffer value (measured by H+ injection) was greatly increased by exposure to CO2 Ringer. Acetazolamide reduced this effect of CO2, suggesting that the function of intracellular carbonic anhydrase may be to maximize the internal buffering power in CO2. 9. It was concluded that the internal HCO3- was determined primarily by the CO2 level and pHi, that internal HCO3- made a large contribution to the buffering power, and that after internal acidfication pHi was restored to normal by active transport of H+, OH- or HCO3- across the cell membrane. The active transport was much faster in CO2 than in CO2-free Ringer.

1. Voltage-clamp experiments were carried out in calf Purkinje fibres to determine the basis of transient depolarizations (TDs) associated with digitalis-induced arrhythmias. 2. Under the influence of strophanthidin, depolarizing clamp pulses were followed by a transient inward current (TI) which was small or absent in untreated preparations. The TI also appeared in the wake of a train of action potentials. 8. The TI can help generate spontaneous depolarizations in preparations showing the low voltage oscillations which often occur with advanced digitalis toxicity. 8. The TI can help generate spontaneous depolarizations in preparations showing the 'low voltage oscillation' which often occur with advanced digitais toxicity. It was designated TI because its magnitude and timing were appropriate to account for the TD. 3. Longitudinal voltage non-uniformity during the TI was determined with two voltage-recording micro-electrodes. Although the non-uniformity was not severe, the TI wave form was observed when the voltage difference signal was used to measure membrane current density. 4. Over the diastolic range of potential, the strophanthidin-induced TI appeared superimposed upon the normal pace-maker mechanism, the decay of a potassium current, iK2. The TI could be dissociated from iK2, however, by means of its unusual kinetic properties. 5. TIs could also be recorded at holding potentials positive to -55 mV, i.e. outside the range where iK2 deactivation occurs. 6. The TI amplitude showed a slow and strongly sigmoid dependence on the duration of the preceding depolarizing pulse. Stronger depolarizing reduced the TI amplitude, while slowing and exaggerating the sigmoid time-dependence. 7. Two clamp pulses in close succession gave additive effects in evoking a subsequent TI. This finding and the sigmoid time-dependence fit with previous observations that TDs are most prominent following a series of closely spaced action potentials.

Vibration threshold determinations were made by means of an electromagnetic vibrator at three sites (carpal, tibial, and tarsal), which were primarily selected for examining patients with polyneuropathy. Because of the vast variation demonstrated for both vibrator output and tissue damping, the thresholds were expressed in terms of amplitude of stimulator movement measured by means of an accelerometer, instead of applied voltage which is commonly used. Statistical analysis revealed a higher power of discimination for amplitude measurements at all three stimulus sites. Digital read-out gave the best statistical result and was also most practical. Reference values obtained from 110 healthy males, 10 to 74 years of age, were highly correlated with age for both upper and lower extremities. The variance of the vibration perception threshold was less than that of the disappearance threshold, and determination of the perception threshold alone may be sufficient in most cases.

We assessed 882 patients presenting with a proximal femoral fracture by a new mobility score and by a mental test score, to determine which was of the most value in forecasting mortality at one year. Both scores gave a highly significant prediction, but the mobility score had a greater predictive value and is easier to perform.

A bacterial restriction endonuclease has been used to produce specific fragments of SV40 DNA. Digestion of DNA from plaque-purified stocks of SV40 with the restriction endonuclease from Hemophilus influenzae gave 11 fragments resolvable by polyacrylamide gel electrophoresis, eight of which were equimolar with the original DNA. The fragments ranged from about 6.5 x 10(5) to 7.4 x 10(4) daltons, as determined by electron microscopy, DNA content, or electrophoretic mobility.

Previous studies have shown that lymph node (LN) T cells from mice given repeated injections of anti-mu antisera from birth (mu sm) fail to mount secondary T proliferative responses to antigen in vitro after s.c. priming in vivo. This finding raised the possibility that priming of T cells in LN depends on the presence of B cells, Ig+ B lymphocytes being absent in mu sm. In support of this idea, the present paper shows that the priming defect in LN of mu sm can be largely overcome by injecting B cell populations s.c. 1 day before s.c. priming with antigen. Restoration of LN priming was observed with s.c. injection of highly purified populations of small B cells but not with heat-killed or lightly irradiated B cells. Homing studies indicated that approximately 10% of s.c.-injected B cells reached the draining LN. In other studies, irradiated mice injected i.v. with purified T cells manifested poor priming in LN after s.c. injection of antigen. It was reasoned that the LN priming defect in this situation reflected the lack of B cells in irradiated mice, B cells being highly radiosensitive. In support of this notion, it was found that s.c. injection of B cells into irradiated recipients of T cells led to high priming of T cells in LN after s.c. injection of antigen. Although T cells exposed to antigen in B-depleted LN of mu sm and irradiated mice gave negligible T proliferative responses in vitro, low but significant levels of primed T helper function were detected in a sensitive T helper assay in vivo. In light of this finding, our working hypothesis is that the initial induction of T cells to antigen in LN is controlled by resident dendritic cells (or other non-B antigen-presenting cells), the main role of B cells being to control the clonal expansion of activated T cells.

BACKGROUND

Cardiovascular mortality is extremely high in end-stage renal disease. Cardiovascular mortality risk also is increased in selected (high-risk) individuals with mild to moderate impairment of renal function. It is not clear whether a similar association exists in the general population and, if so, through what mechanisms. We investigated the association of renal function with all-cause and cardiovascular mortality in a population-based cohort and explored potential mechanisms underlying any such relationship.

METHODS

An age-, sex-, and glucose-tolerance-stratified sample (N = 631) of a population-based cohort aged 50 to 75 years was followed prospectively. After up to 10.2 years of follow-up, 117 subjects had died (50 of cardiovascular causes). At baseline, renal function was estimated by the serum creatinine level, the Cockcroft-Gault formula and Levey's equation.

RESULTS

At baseline, the mean age was 64 +/- 7 years, 48% were men, 55% had hypertension, and 27% (by design) had type 2 diabetes. Serum creatinine was 91.7 +/- 19.0 micromol/L; creatinine clearance as estimated by the Cockroft-Gault formula was 72.5 +/- 13.7 mL/min/1.73 m(2), and the glomerular filtration rate (GFR) estimated by Levey's equation was 67.8 +/- 12.1 mL/min/1.73 m(2). Renal function was inversely associated with all-cause and with cardiovascular mortality. Relative risks (95% confidence intervals) were 1.08 (1.04 to 1.13) and 1.11 (1.07 to 1.16) per 5 micromol/L increase of serum creatinine; 1.07 (0.98 to 1.17) and 1.15 (1.01 to 1.31) for each decrease of 5 mL/min/1.73 m(2) creatinine clearance; and 1.15 (1.05 to 1.26) and 1.26 (1.12 to 1.42) for each decrease of 5 mL/min/1.73 m(2) of GFR. These associations remained after adjusting for age, sex, glucose tolerance status, hypertension, prior cardiovascular disease, low-density lipoprotein cholesterol, homocysteine, (micro)albuminuria, von Willebrand factor, soluble vascular adhesion molecule-1 and C-reactive protein. Analyses in diabetic and hypertensive subjects gave similar results.

CONCLUSIONS

Mild to moderate loss of renal function is strongly associated with an increased risk of cardiovascular mortality. The mechanism behind this association is unclear but does not appear to involve common risk factors such as hypertension, diabetes or hyperhomocysteinemia. Estimation of renal function by relatively simple methods therefore may be a valuable tool for cardiovascular risk assessment over and above that provided by conventional risk factors. Our results were obtained in a general middle-aged to elderly population, and thus have broad applicability.

OBJECTIVE

Perturbations to the prenatal environment have been associated with the development of adult chronic disease, findings that gave rise to the "Barker Hypothesis" or the "developmental origins of adult disease" concept. In this study, we used an animal model to determine the metabolic consequences of maternal prenatal stress and high-fat feeding on the developing offspring.

METHODS

Pregnant female Sprague-Dawley rats were maintained on standard chow or 60% high-fat diet throughout gestation and lactation. Half of each group were exposed to a novel variable stress paradigm during the 3rd week of gestation, whereas control dams were left undisturbed. Body weight, body composition, glucose tolerance, and endocrine parameters were measured in offspring through early adulthood.

RESULTS

Male and female pups from dams that experienced prenatal stress and/or were on a high-fat diet weighed more beginning on postnatal day 7 compared with standard chow-control pups. Access to high-fat diet at weaning increased the body weight effect through early adulthood and was attributable to greater adiposity. Pups weaned onto standard chow diet showed no significant difference in glucose clearance or insulin secretion. However, pups weaned onto high-fat diet had impaired glucose tolerance if their dams were on a high-fat diet, experienced prenatal stress, or both.

CONCLUSIONS

Our data demonstrate that prenatal stress and/or high-fat diet during the intrauterine or postnatal environment affects offspring in a manner that increases their susceptibility to diet-induced obesity and leads to secondary adverse metabolic consequences.

1. Post-spike facilitation of e.m.g. activity by monkey motor cortex neurones has been investigated in different hand and forearm muscles. 2. Seventy-eight neurones were recorded concurrently with between five and ten different muscles. Forty-seven neurones were identified as cortico-motor by the presence of post-spike facilitation in the spike-triggered average of at least one of the tested muscles. 3. All forty-seven cortico-motor neurones showed clear increases in activity during performance of a precision grip task by the monkey, and all of them were co-activated with the sampled muscles. 4. To assess the divergence of facilitation from a single cortico-motor neurone to different muscles, spike-triggered averages were constructed with all of the concurrently recorded muscles. The number of muscles in the sample, and the number of muscles showing post-spike facilitation, were corrected by excluding any post-spike facilitation which could have arisen by cross-talk between the different pairs of e.m.g. electrodes. 5. Most cortico-motor neurones produced post-spike facilitation in a restricted number of tested muscles. The mean number of post-spike facilitation-bearing muscles per cortico-motor cell rose from 1.4 +/- 0.5 (S.D.) when five muscles were sampled to 2.0 +/- 1.5 when ten were sampled. On average, each cortico-motor neurone produced post-spike facilitation in 27% of the tested muscles. Only three of forty-seven cortico-motor neurones gave post-spike facilitation in half or more of the tested muscles. 6. The distribution pattern of post-spike facilitation among the muscles sampled with a given cortico-motor neurone was not altered when the spike-triggered averages were constructed from cortico-motor cell and e.m.g. activity recorded during two different phases of the precision grip task, or during performance of a quite different, power grip, task. 7. Cortico-motor cells which produced post-spike facilitation in two or more different muscles often did so in muscles with synergistic functions. 8. It is suggested that cortico-motor neurones may contribute to relatively independent finger movements by virtue of their selective facilitation of hand muscles leading to a fractionated pattern of muscle activity.

The inhibitory activity of tea against tumorigenesis has been demonstrated in many animal models and has been suggested by some epidemiological studies. Such activity has generally been attributed to tea catechins. To understand the bioavailability of tea catechins in humans, we gave 18 individuals different amounts of green tea and measured the time-dependent plasma concentrations and urinary excretion of tea catechins. After taking 1.5, 3.0, and 4.5 g of decaffeinated green tea solids (dissolved in 500 ml of water), the maximum plasma concentration (Cmax) of (-)-epigallocatechin-3-gallate (EGCG) was 326 ng/ml, the Cmax of (-)-epigallocatechin (EGC) was 550 ng/ml, and the Cmax of (-)-epicatechin (EC) was 190 ng/ml. These Cmax values were observed at 1.4-2.4 h after ingestion of the tea preparation. When the dosage was increased from 1.5 to 3.0 g, the Cmax values increased 2.7-3.4-fold, but increasing the dose to 4.5 g did not increase the Cmax values significantly, which suggested a saturation phenomenon. The half-life of EGCG (5.0-5.5 h) seemed to be higher than the half-life of EGC or EC (2.5-3.4 h). EGC and EC, but not EGCG, were excreted in the urine. Over 90% of the total urinary EGC and EC was excreted within 8 h. When the tea dosage was increased, the amount of EGC and EC excretion seemed to increase, but a clear dose-response relationship was not observed. The present study provides basic pharmacokinetic parameters of green tea catechins in humans; these parameters may be used to estimate the levels of these compounds after drinking tea.

An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.