In situ hybridization analysis of mouse embryos shows the seven members of the Hox-2 complex to be differentially expressed in the central and peripheral nervous system and in mesodermal derivatives (somites and lung). Beginning at the 5' end of the cluster, each successive gene displays a more anterior boundary of expression in the central nervous system. A gene's position in the Hox-2 cluster therefore reflects its relative domain of expression along the anteroposterior axis of the embryo, a feature observed with Drosophila homeotic genes. Sequence comparisons of the Hox-2 cluster with other mouse and Drosophila homeobox genes have defined subgroups of related genes; in the mouse there are four clusters related by duplication and divergence. Alignment shows a clear relationship among genes in the mouse and Drosophila complexes, based on relative position, sequence identity, and domains of expression along the rostral-caudal axis. Our results argue that these complexes arose from a common ancestor, present before the divergence of lineages that gave rise to arthropods and vertebrates.

The complete sequencing of bacterial genomes has revealed a large number of drug transporter genes. In Escherichia coli, there are 37 open reading frames (ORFs) assumed to be drug transporter genes on the basis of sequence similarities, although the transport capabilities of most of them have not been established yet. We cloned all 37 putative drug transporter genes in E. coli and investigated their drug resistance phenotypes using an E. coli drug-sensitive mutant as a host. E. coli cells transformed with a plasmid carrying one of 20 ORFs, i.e., fsr, mdfA, yceE, yceL, bcr, emrKY, emrAB, emrD, yidY, yjiO, ydhE, acrAB, cusA (formerly ybdE), yegMNO, acrD, acrEF, yhiUV, emrE, ydgFE, and ybjYZ, exhibited increased resistance to some of the 26 representative antimicrobial agents and chemical compounds tested in this study. Of these 20 ORFs, cusA, yegMNO, ydgFE, yceE, yceL, yidY, and ybjYZ are novel drug resistance genes. The fsr, bcr, yjiO, ydhE, acrD, and yhiUV genes gave broader resistance spectra than previously reported.

The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations in nuclear NF-kappaB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-alpha at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-kappaB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-kappaB-dependent gene expression, which supports the idea that oscillation frequency has a functional role.

To develop and validate a radiomics nomogram for preoperative prediction of lymph node (LN) metastasis in patients with colorectal cancer (CRC).

The prediction model was developed in a primary cohort that consisted of 326 patients with clinicopathologically confirmed CRC, and data was gathered from January 2007 to April 2010. Radiomic features were extracted from portal venous-phase computed tomography (CT) of CRC. Lasso regression model was used for data dimension reduction, feature selection, and radiomics signature building. Multivariable logistic regression analysis was used to develop the predicting model, we incorporated the radiomics signature, CT-reported LN status, and independent clinicopathologic risk factors, and this was presented with a radiomics nomogram. The performance of the nomogram was assessed with respect to its calibration, discrimination, and clinical usefulness. Internal validation was assessed. An independent validation cohort contained 200 consecutive patients from May 2010 to December 2011.

The radiomics signature, which consisted of 24 selected features, was significantly associated with LN status (P < .001 for both primary and validation cohorts). Predictors contained in the individualized prediction nomogram included the radiomics signature, CT-reported LN status, and carcinoembryonic antigen level. Addition of histologic grade to the nomogram failed to show incremental prognostic value. The model showed good discrimination, with a C-index of 0.736 (C-index, 0.759 and 0.766 through internal validation), and good calibration. Application of the nomogram in the validation cohort still gave good discrimination (C-index, 0.778 [95% CI, 0.769 to 0.787]) and good calibration. Decision curve analysis demonstrated that the radiomics nomogram was clinically useful.

This study presents a radiomics nomogram that incorporates the radiomics signature, CT-reported LN status, and clinical risk factors, which can be conveniently used to facilitate the preoperative individualized prediction of LN metastasis in patients with CRC.

BACKGROUND

Most patients who have active Crohn's disease are treated initially with corticosteroids. Although this approach usually controls symptoms, many patients become resistant to or dependent on corticosteroids, and long exposure is associated with an increased risk of mortality. We aimed to compare the effectiveness of early use of combined immunosuppression with conventional management in patients with active Crohn's disease who had not previously received glucocorticoids, antimetabolites, or infliximab.

METHODS

We did a 2-year open-label randomised trial at 18 centres in Belgium, Holland, and Germany between May, 2001, and January, 2004. We randomly assigned 133 patients to either early combined immunosuppression or conventional treatment. The 67 patients assigned to combined immunosuppression received three infusions of infliximab (5 mg/kg of bodyweight) at weeks 0, 2, and 6, with azathioprine. We gave additional treatment with infliximab and, if necessary, corticosteroids, to control disease activity. 66 patients assigned to conventional management received corticosteroids, followed, in sequence, by azathioprine and infliximab. The primary outcome measures were remission without corticosteroids and without bowel resection at weeks 26 and 52. Analysis was by modified intention to treat. This trial was registered with ClinicalTrials.gov, number NCT00554710.

RESULTS

Four patients (two in each group) did not receive treatment as per protocol. At week 26, 39 (60.0%) of 65 patients in the combined immunosuppression group were in remission without corticosteroids and without surgical resection, compared with 23 (35.9%) of 64 controls, for an absolute difference of 24.1% (95% CI 7.3-40.8, p=0.0062). Corresponding rates at week 52 were 40/65 (61.5%) and 27/64 (42.2%) (absolute difference 19.3%, 95% CI 2.4-36.3, p=0.0278). 20 of the 65 patients (30.8%) in the early combined immunosuppression group had serious adverse events, compared with 19 of 64 (25.3%) controls (p=1.0).

CONCLUSIONS

Combined immunosuppression was more effective than conventional management for induction of remission and reduction of corticosteroid use in patients who had been recently diagnosed with Crohn's disease. Initiation of more intensive treatment early in the course of the disease could result in better outcomes.

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations >> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).

Peanut agglutinin was purified by affinity chromatography on Sepharose-epsilon-aminocaproyl-beta-D-galactopyranosylamine. The purified lectin obtained in a yield of 150 mg/100 g of defatted peanut was homogeneous on polyacrylamide gel electrophoresis, ultracentrifugation, and gel filtration. This intrinsic sedimentation coefficient (So20,w) and the intrinsic diffusion coefficient (Do20,w) were estimated at pH 7.4 as 5.7 +/- 0.1 S and 5.0 X 10(-7) cm2s(-1), respectively. The molecular weight of the agglutinin, determined by sedimentation and diffusion and by gel filtration, was found to be 110,000. Disc gel electrophoresis and gel filtration, both in the presence of sodium dodecyl sulfate, gave a single component of Mr = 27,500 suggesting that the lectin is a tetramer composed of four subunits. Four alanine residues per 110,000 g were found by NH2-terminal analysis and the sequence of the five NH2-terminal amino acids was: ALa-Glu-Ser-Val-Thr. Each cycle in a sequenator gave a single amino acid, suggesting that the four subunits are identical. Peanut agglutinin does not contain covalently bound sugar; it is devoid of cysteine and cystine, low in methionine, histidine, and tryptophan, but rich in acidic and hydroxyamino acids. The lectin agglutinated erthrocytes of human ABO blood types equally well, but only after they have been treated with neuraminidase. Of the monosaccharides tested for inhibition of hemagglutination only D-galactose and alpha- and beta-D-galactosides were active. High inhibitory activity was found with the Discaccharide DGalbeta(1 in equilibrium 3)DGalNAc and with the disialylated glycoproteins: alpha1-acid glycoprotein, fetuin, glycophorin, and human blood group NN or MM antigen. These desialylated glycoproteins also reacted with the lectin to form precipitin bands in Ouchterlony double diffusion in agar.

Endogenous mechanisms that act in the resolution of acute inflammation are essential for host defense and the return to homeostasis. Resolvin D1 (RvD1), biosynthesized during resolution, displays potent and stereoselective anti-inflammatory actions, such as limiting neutrophil infiltration and proresolving actions. Here, we demonstrate that RvD1 actions on human polymorphonuclear leukocytes (PMNs) are pertussis toxin sensitive, decrease actin polymerization, and block LTB(4)-regulated adhesion molecules (beta2 integrins). Synthetic [(3)H]-RvD1 was prepared, which revealed specific RvD1 recognition sites on human leukocytes. Screening systems to identify receptors for RvD1 gave two candidates--ALX, a lipoxin A(4) receptor, and GPR32, an orphan--that were confirmed using a beta-arrestin-based ligand receptor system. Nuclear receptors including retinoid X receptor-alpha and peroxisome proliferator-activated receptor-alpha, -delta, -gamma were not activated by either resolvin E1 or RvD1 at bioactive nanomolar concentrations. RvD1 enhanced macrophage phagocytosis of zymosan and apoptotic PMNs, which increased with overexpression of human ALX and GPR32 and decreased with selective knockdown of these G-protein-coupled receptors. Also, ALX and GPR32 surface expression in human monocytes was up-regulated by zymosan and granulocyte-monocyte-colony-stimulating factor. These results indicate that RvD1 specifically interacts with both ALX and GPR32 on phagocytes and suggest that each plays a role in resolving acute inflammation.

Glial fibrillary acidic protein (GFAP) is the main intermediate filament protein in mature astrocytes, but also an important component of the cytoskeleton in astrocytes during development. Major recent developments in astrocyte biology and the discovery of novel intermediate filament functions enticed the interest in the function of GFAP. The discovery of various GFAP splice variants gave an additional boost to explore this protein in more detail. The structural role of GFAP in astrocytes has been widely accepted for a long time, but over the years, GFAP has been shown to be involved in astrocyte functions, which are important during regeneration, synaptic plasticity and reactive gliosis. Moreover, different subpopulations of astrocytes have been identified, which are likely to have distinctive tasks in brain physiology and pathology, and which are not only classified by their spatial and temporal appearance, but also by their specific expression of intermediate filaments, including distinct GFAP isoforms. The presence of these isoforms enhances the complexity of the astrocyte cytoskeleton and is likely to underlie subtype specific functions. In this review we discuss the versatility of the GFAP cytoskeletal network from gene to function with a focus on astrocytes during human brain development, aging and disease.

Genetic screens in Drosophila have lead to the discovery of many genes important for patterning and signal transduction in diverse organisms. Traditionally, the phenotypic effects of loss-of-function mutations are analyzed. As an alternative way to link genes and function, I have developed a versatile misexpression screen in Drosophila, the first such screen in higher eukaryotes. The screen identifies genes that, when over- or misexpressed in a pattern of interest, give a specific phenotype or modulate an existing mutant phenotype. It is based on Gal4 transactivation of a mobile enhancer and promoter that "targets" random endogenous genes for expression. The modular design of the screen allows directed expression in any temporal or spatial pattern. When activated in the developing eye, 4% of target inserts gave dominant phenotypes. One insertion was in the gene encoding Ras GTPase-activating protein; its overexpression phenotype was strongly enhanced by a mutation in Ras1. Thus, biologically relevant phenotypes and genetic interactions are identified using this method. The screen is a powerful new tool for developmental genetics; similar approaches can also be developed for other organisms.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg-sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.

BACKGROUND

Social causation (adversity and stress) vs social selection (downward mobility from familial liability to mental illness) are competing theories about the origins of mental illness.

OBJECTIVE

To test the role of social selection vs social causation of childhood psychopathology using a natural experiment.

METHODS

Quasi-experimental, longitudinal study.

METHODS

A representative population sample of 1420 rural children aged 9 to 13 years at intake were given annual psychiatric assessments for 8 years (1993-2000). One quarter of the sample were American Indian, and the remaining were predominantly white. Halfway through the study, a casino opening on the Indian reservation gave every American Indian an income supplement that increased annually. This increase moved 14% of study families out of poverty, while 53% remained poor, and 32% were never poor. Incomes of non-Indian families were unaffected.

METHODS

Levels of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, psychiatric symptoms in the never-poor, persistently poor, and ex-poor children were compared for the 4 years before and after the casino opened.

RESULTS

Before the casino opened, the persistently poor and ex-poor children had more psychiatric symptoms (4.38 and 4.28, respectively) than the never-poor children (2.75), but after the opening levels among the ex-poor fell to those of the never-poor children, while levels among those who were persistently poor remained high (odds ratio, 1.50; 95% confidence interval, 1.08-2.09; and odds ratio, 0.91; 95% confidence interval, 0.77-1.07, respectively). The effect was specific to symptoms of conduct and oppositional defiant disorders. Anxiety and depression symptoms were unaffected. Similar results were found in non-Indian children whose families moved out of poverty during the same period.

CONCLUSIONS

An income intervention that moved families out of poverty for reasons that cannot be ascribed to family characteristics had a major effect on some types of children's psychiatric disorders, but not on others. Results support a social causation explanation for conduct and oppositional disorder, but not for anxiety or depression.

Logistic regression (LR), discriminant analysis (DA), and neural networks (NN) were used to predict ordered and disordered regions in proteins. Training data were from a set of non-redundant X-ray crystal structures, with the data being partitioned into N-terminal, C-terminal and internal (I) regions. The DA and LR methods gave almost identical 5-cross validation accuracies that averaged to the following values: 75.9 +/- 3.1% (N-regions), 70.7 +/- 1.5% (I-regions), and 74.6 +/- 4.4% (C-regions). NN predictions gave slightly higher scores: 78.8 +/- 1.2% (N-regions), 72.5 +/- 1.2% (I-regions), and 75.3 +/- 3.3% (C-regions). Predictions improved with length of the disordered regions. Averaged over the three methods, values ranged from 52% to 78% for length = 9-14 to>>/= 21, respectively, for I-regions, from 72% to 81% for length = 5 to 12-15, respectively, for N-regions, and from 70% to 80% for length = 5 to 12-15, respectively, for C-regions. These data support the hypothesis that disorder is encoded by the amino acid sequence.

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

N-methyl-D-aspartic acid (NMDA) receptors transiently transfected into mammalian HEK-293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Deactivation time course recorded in response to fast-glutamate pulses were studied in isolated and lifted cells, as well as in outside-out membrane patches excised from cells expressing recombinant NR1 subunits in combination with the NR2A, NR2B, NR2C, or NR2D NMDA receptor subunits. Transfected cells were preidentified by the fluorescence emitted from the coexpressed Aequorea victoria jellyfish Green Lantern protein. Currents generated by NR1/NR2A channels displayed double exponential deactivation time course being faster than that in NR1/NR2B or NR1/NR2C channels. However, a large decay variability was observed within each cotransfection, suggesting that mechanisms additional to subunit composition may also regulate deactivation time course. NR1/NR2D channels displayed slowly deactivating currents. Channel deactivation was fast and comparable among receptors obtained by cotransfecting five distinct spliced variants of the NR1 subunit, each with the NR2A subunit. Additionally, recovery from desensitization was slower for NR1/NR2B than for NR1/NR2A channels. The average deactivation time course of responses to brief L-glutamate applications in cells where NR1/NR2A/NR2B cDNAs were cotransfected at variable ratio was intermediate between those of the NR1/NR2A and NR1/NR2B channels. Although immunocytochemical evidence indicates that the majority of cells are cotransfected by all plasmids in triple transfection, our experimental condition did not allow for a tight control of the expression of NMDA receptor subunits. This produced the result that many cells were characterized by deactivation time course and haloperidol sensitivities of separate NR1/NR2A and NR1/NR2B subunit heteromers. We also speculate on the possible formation of channels resulting from the coassembly in the same receptor of NR1/NR2A/NR2B subunits from a minority of cells that gave responses to brief application of L-glutamate characterized by slow deactivation time course and decreased haloperidol sensitivity.

The continuously proliferating clones L/B AgA2, CB/Bm 7, Ba/C1, and Bc/Bm 11 were established from bone marrow of MRL/LPR, CBA/J, and BALB/c mice. These clones carry the B cell lineage surface antigen B-220 but not antigens normally expressed on mature B lymphocytes, myeloid cells, or T lymphocytes. Their immunoglobulin mu heavy chain and kappa light chain genes are in germ-line configuration. The G418 resistance gene was introduced into each clone with a retrovirus vector and then used as a selective marker for the progeny of transfected cells. Clones L/B AgA2, CB/Bm 7, and Bc/Bm 11, but not Ba/C1, could develop into antibody-secreting cells after in vivo transfer. None gave rise to cells responsive to polyclonal T cell activators, nor did any differentiate into cells that could develop into granulocyte/macrophage-colony-forming cells in vitro. All grew in interleukin 3 but not in other cytokines. We conclude that clones L/B AgA2, CB/Bm 7, and Bc/Bm 11 are early precursors of B lymphocytes.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

C3b inactivator accelerator (A-C3bINA) was isolated from human plasma. An antiserum produced against the purified protein gave a reaction of identity with beta 1 H, a well-documented contaminant of C3 preparations. Beta 1 H appears to be composed of a single polypeptide chain containing a significant quantity of carbohydrate, and having a sedimentation coefficient of 5.6 on analytical, and 6.4 on sucrose density gradient ultracentrifugation. Its mol wt based on SDS polyacrylamide gel electrophoresis and equilibrium sedimentation is approximately 150,000, whereas it elutes from Sephadex G200 with an apparent mol wt of 300,000, suggesting that beta 1 H is an asymmetric molecule. Beta 1 H potentiates the inactivation of C3b by C3b inactivator, binds to EAC43 to limit the formation of EAC43bB and EAC43bBP, and in contrast to C3b inactivator, it increases the rate of loss of hemolytic sites from EAC43bB and EAC43bBP. For the C3b inactivator-potentiating effect, beta 1 H and C3b inactivator must necessarily be simultaneously present. The kinetics of inactivation of C3b by C3b inactivator and beta 1 H are first order, suggesting that potentiation is not a multistep process. The mechanisms of binding to C3b and inhibition of the alternative pathway convertases C3bB and C3bBP are currently unknown.

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.

BACKGROUND

The skin is responsible for forming a variety of epidermal structures that differ amongst vertebrates. In each case the specific structure (for example scale, feather or hair) arises from an epidermal placode as a result of epithelial-mesenchymal interactions with the underlying dermal mesenchyme. Expression of members of the Wnt, Hedgehog and bone morphogenetic protein families (Wnt10b, Sonic hedgehog (Shh) and Bmp2/Bmp4, respectively) in the epidermis correlates with the initiation of hair follicle formation. Further, their expression continues into either the epidermally derived hair matrix which forms the hair itself, or the dermal papilla which is responsible for induction of the hair matrix. To address the role of Shh in the hair follicle, we have examined Shh null mutant mice.

RESULTS

We found that follicle development in the Shh mutant embryo arrested after the initial epidermal-dermal interactions that lead to the formation of a dermal papilla anlage and ingrowth of the epidermis. Wnt10b, Bmp2 and Bmp4 continued to be expressed at this time, however. When grafted to nude mice (which lack T cells), Shh mutant skin gave rise to large abnormal follicles containing a small dermal papilla. Although these follicles showed high rates of proliferation and some differentiation of hair matrix cells into hair-shaft-like material, no hair was formed.

CONCLUSIONS

Shh signaling is not required for initiating hair follicle development. Shh signaling is essential, however, for controlling ingrowth and morphogenesis of the hair follicle.

A weight-height index of adiposity should indicate the relative fatness of subjects of differing height unless obesity is itself correlated with height. The average body fat among adult women attending a hospital outpatient clinic for obesity was 40.5 percent of body weight. The height of an unselected series of 286 of these outpatients was found to be similar to that of the general population of women of similar age, which indicates that obesity in adult women is not significantly related to height. Body composition was measured by body density, body water and body potassium in a series of 104 female and 24 male subjects aged 14-60 years. In both sexes density, water and potassium gave progressively higher estimates of body fat (kg), and there was a significant difference between the values by different methods. The average of the estimates by these three methods was taken to be the 'true' value for each individual (F kg). Regression of F/H2 on W/H2 (Quetelet's index) gave a correlation coefficient of 0.955 for women and 0.943 for men. The deviation of the body fat estimated from Quetelet's formula from the 'true' value was not much greater than that when density, water or potassium were used as a basis for estimating body fat. It is concluded that Quetelet's formula is both a convenient and reliable indicator of obesity.

Selectins tether to the blood vessel wall leukocytes that are flowing in the bloodstream and support subsequent labile rolling interactions as the leukocytes are subjected to hydrodynamic drag forces. To support this rolling, selectins have been proposed to have rapid bond association and dissociation rate constants, and special mechanical properties linking tensile forces and bond dissociation. We have visualized transient tethering and release of neutrophils in hydrodynamic flow on lipid bilayers containing densities of P-selectin below those required to support rolling. We report here that transient tethers had first-order kinetics and other characteristics suggesting a unimolecular interaction between P-selectin and its glycoprotein ligand (PSGL-1). The unstressed dissociation constant (off rate) was 1 s-1. Hydrodynamic shear stresses of up to 1.1 dyn cm-2, corresponding to a force on the bond of up to 110 pN, increased the off rate only modestly, to 3.5 s-1. The data was adequately matched by a proposed equation relating off rate to the exponential of tensile force on the bond and the bond interaction distance, and gave a bond interaction distance of 0.5 A. This distance is compatible with hydrogen and metal coordination bonds between P-selectin and PSGL-1. Fast on and off rates, together with the high tensile strength of the selectin bond, appear necessary to support rolling at physiological shear stresses.

BACKGROUND

The relevance to coronary heart disease (CHD) of cytokines that govern inflammatory cascades, such as interleukin-6 (IL-6), may be underestimated because such mediators are short acting and prone to fluctuations. We evaluated associations of long-term circulating IL-6 levels with CHD risk (defined as nonfatal myocardial infarction [MI] or fatal CHD) in two population-based cohorts, involving serial measurements to enable correction for within-person variability. We updated a systematic review to put the new findings in context.

RESULTS

Measurements were made in samples obtained at baseline from 2,138 patients who had a first-ever nonfatal MI or died of CHD during follow-up, and from 4,267 controls in two cohorts comprising 24,230 participants. Correction for within-person variability was made using data from repeat measurements taken several years apart in several hundred participants. The year-to-year variability of IL-6 values within individuals was relatively high (regression dilution ratios of 0.41, 95% confidence interval [CI] 0.28-0.53, over 4 y, and 0.35, 95% CI 0.23-0.48, over 12 y). Ignoring this variability, we found an odds ratio for CHD, adjusted for several established risk factors, of 1.46 (95% CI 1.29-1.65) per 2 standard deviation (SD) increase of baseline IL-6 values, similar to that for baseline C-reactive protein. After correction for within-person variability, the odds ratio for CHD was 2.14 (95% CI 1.45-3.15) with long-term average ("usual") IL-6, similar to those for some established risk factors. Increasing IL-6 levels were associated with progressively increasing CHD risk. An updated systematic review of electronic databases and other sources identified 15 relevant previous population-based prospective studies of IL-6 and clinical coronary outcomes (i.e., MI or coronary death). Including the two current studies, the 17 available prospective studies gave a combined odds ratio of 1.61 (95% CI 1.42-1.83) per 2 SD increase in baseline IL-6 (corresponding to an odds ratio of 3.34 [95% CI 2.45-4.56] per 2 SD increase in usual [long-term average] IL-6 levels).

CONCLUSIONS

Long-term IL-6 levels are associated with CHD risk about as strongly as are some major established risk factors, but causality remains uncertain. These findings highlight the potential relevance of IL-6-mediated pathways to CHD.

Agar diffusion techniques are used widely to assay plant extracts for antimicrobial activity, but there are problems associated with this technique. A micro-dilution technique was developed using 96-well microplates and tetrazolium salts to indicate bacterial growth. p-Iodonitrotetrazolium violet [0.2 mg/ml] gave better results than tetrazolium red or thiazolyl blue. The method is quick, worked well with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli and with non-aqueous extracts from many different plants. The method gave reproducible results; required only 10-25 microliters of extract to determine minimal inhibitory concentrations, distinguished between microcidal and microstatic effects, and provided a permanent record of the results. Using S. aureus, and a Combretum molle extract, the technique was 32 times more sensitive than agar diffusion techniques and was not sensitive to culture age of the test organism up to 24 hours. The S. aureus culture could be stored up to 10 days in a cold room with little effect on the assay results. This method was useful in screening plants for antimicrobial activity and for the bioassay-guided isolation of antimicrobial compounds from plants. MIC values determined for sulfisoxazole, norfloxacin, gentamicin, and nitrofuratoin were similar to values indicated in the literature but values obtained with trimethroprim and ampicillin were higher with some bacteria.