Trace metals as biomarkers for eumelanin pigment in the fossil record.

Well-preserved fossils of pivotal early bird and nonavian theropod species have provided unequivocal evidence for feathers and/or downlike integuments. Recent studies have reconstructed color on the basis of melanosome structure; however, the chemistry of these proposed melanosomes has remained unknown. We applied synchrotron x-ray techniques to several fossil and extant organisms, including Confuciusornis sanctus, in order to map and characterize possible chemical residues of melanin pigments. Results show that trace metals, such as copper, are present in fossils as organometallic compounds most likely derived from original eumelanin. The distribution of these compounds provides a long-lived biomarker of melanin presence and density within a range of fossilized organisms. Metal zoning patterns may be preserved long after melanosome structures have been destroyed.

A spatial and seasonal assessment of river water chemistry across North West England.

This paper presents information on the spatial and seasonal patterns of river water chemistry at approximately 800 sites in North West England based on data from the Environment Agency regional monitoring programme. Within a GIS framework, the linkages between average water chemistry (pH, sulphate, base cations, nutrients and metals) catchment characteristics (topography, land cover, soil hydrology, base flow index and geology), rainfall, deposition chemistry and geo-spatial information on discharge consents (point sources) are examined. Water quality maps reveal that there is a clear distinction between the uplands and lowlands. Upland waters are acidic and have low concentrations of base cations, explained by background geological sources and land cover. Localised high concentrations of metals occur in areas of the Cumbrian Fells which are subjected to mining effluent inputs. Nutrient concentrations are low in the uplands with the exception sites receiving effluent inputs from rural point sources. In the lowlands, both past and present human activities have a major impact on river water chemistry, especially in the urban and industrial heartlands of Greater Manchester, south Lancashire and Merseyside. Over 40% of the sites have average orthophosphate concentrations >0.1mg-Pl(-1). Results suggest that the dominant control on orthophosphate concentrations is point source contributions from sewage effluent inputs. Diffuse agricultural sources are also important, although this influence is masked by the impact of point sources. Average nitrate concentrations are linked to the coverage of arable land, although sewage effluent inputs have a significant effect on nitrate concentrations. Metal concentrations in the lowlands are linked to diffuse and point sources. The study demonstrates that point sources, as well as diffuse sources, need to be considered when targeting measures for the effective reduction in river nutrient concentrations. This issue is clearly important with regards to the European Union Water Framework Directive, eutrophication and river water quality.

CD38 as a prognostic marker in chronic lymphocytic leukaemia at a single New Zealand centre: patient survival in comparison to age- and sex-matched population data.

AIM: The aim of this study is to determine whether the analysis of CD38 expression by chronic lymphocytic leukaemia (CLL) cells provides useful additional prognostic information. METHODS: Clinical, laboratory, overall survival (OS) and treatment-free survival (TFS) data were collected on 130 CLL patients who had CD38 expression analysed at Canterbury Health Laboratories, New Zealand (NZ) during 1998-2008. RESULTS: The detection of any level of CD38 expression by CLL cells was associated with a significantly shorter OS and TFS. When analysis was restricted to Binet stage A patients, CD38 expression identified a subset of patients (21%) who, in common with Binet stage B/C patients, had a significantly shorter OS and TFS (P<0.0015), and a TFS at 4 years of <10%. In contrast, CD38-negative Binet stage A patients had an OS that was not significantly different from that of an age/sex-matched NZ population and a 5-year TFS of 77%. CONCLUSION: This study indicates that, when combined with clinical staging, the presence of any detectable CD38 expression can be used to further improve the identification of CLL patients with more aggressive disease (i.e. Binet stage B/C or Binet stage A and CD38 positive). This will allow better identification of those patients requiring more intensive monitoring and also allow improved patient counselling regarding prognosis.

Arsenic retention and release in ombrotrophic peatlands.

Organic matter can play an important role in the mobility and fate of As in the environment, but there is a lack of data on As biogeochemistry in ombrotrophic peatlands. The aim of this study was to investigate As retention and release in atmospherically contaminated ombrotrophic peat soils in the Peak District National Park (UK). Solid phase As concentrations in the peat soils exceed 25 mg kg(-1). Solid phase As and Fe concentrations are closely correlated at sites where the peat is subjected to drying and oxic conditions. In a wetter zone of the bog, solid phase As and Fe distributions are decoupled, suggesting that As retention in these systems is not solely controlled by the presence of Fe oxides. Comparison of solid phase As and Pb distributions reveals that As has been subjected to post-depositional mobility in areas of water table fluctuation. Conversely, at permanently waterlogged locations As is immobile. Detailed stream water sampling reveals that As is released from the organic-rich uplands soils into the fluvial system. Dissolved As concentrations are highly variable, with values ranging from 0.20 to 7.28 microg l(-1). Stream water As concentrations are elevated during late summer stormflow periods when there has been re-wetting of the peat after significant water table draw-down. Dissolved As is strongly correlated to dissolved organic carbon under stormflow and baseflow. The results of this study suggest that organic matter plays an important role in As dynamics in ombrotrophic peatlands, but further work is needed to identify the exact As binding and release mechanisms. Drying and re-wetting of ombrotrophic peat soils and associated changes in redox status has the potential to lead to increased As mobility. Further work is needed to provide information on how predicted climate change will influence As cycling at sites containing a legacy of atmospheric contamination.

Polycarboxylates inhibit the glucan-binding lectin of Streptococcus sobrinus.

Polycarboxylates, such as carboxymethylcellulose and hyaluronan, were found to be reversible inhibitors of the glucan-binding lectin of Streptococcus sobrinus. When the carboxylate groups were coupled to ethylenediamine, or reduced with carbodiimide-borohydride, inhibitory powers were lost. Similarly, N-deacetylated hyaluronan had poor inhibitory powers, probably due to the introduction of positive charges into the polymer. Other polymers, such as chondroitin sulfates, dextran sulfate, fetuin, heparin were not inhibitors. It appears that inhibition is based on repeating carboxylates, free of influence from ammonium groups. Such polymers have the property of complexing with metals. Earlier studies had concluded that the streptococcal lectin depended on manganese for activity. It is likely the carboxymethylcellulose and hyaluronan perturb essential metal coordination centers in the lectin. Polycarboxylates may have value in oral health care by acting on glucan-dependent microbial adhesion and biofilm formation.

Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin.

The conformational features of dihydrosphingomyelin (DHSM), the major phospholipid of human lens membranes, were investigated by 1H and 31P nuclear magnetic resonance spectroscopy. Several postulates emerge from the observed trends: (a) in partially hydrated samples of DHSM in CDCl3 above 13 mM, at which lipid-lipid interactions prevail, the amide proton is mostly involved in intermolecular H-bonds that link neighboring phospholipids through bridging water molecules. In the absence of water, the NH group is involved in an intramolecular H-bond that restricts the mobility of the phosphate group. (b) In the monomeric form of the lipid molecule, the amide proton of the major conformer is bound intramolecularly with one of the anionic and/or ester oxygens of the phosphate group. A minor conformer may also be present in which the NH proton participates in an intramolecular H-bond linking to the OH group of the sphingoid base. (c) Complete hydration leads to an extension of the head group as water molecules bind to the phosphate and NH groups via H-bonds, thus disrupting the intramolecular H-bonds prevalent at low concentrations.

Conformational studies of sphingolipids by NMR spectroscopy. II. Sphingomyelin.

Sphingomyelin (SM) is the most prevalent sphingolipid in the majority of mammalian membranes. Proton and 31P nuclear magnetic resonance spectral data were acquired to establish the nature of intra- and intermolecular H-bonds in the monomeric and aggregated forms of SM and to assess possible differences between this lipid and dihydrosphingomyelin (DHSM), which lacks the double bond between carbons 4 and 5 of the sphingoid base. The spectral trends suggest the formation of an intramolecular H-bond between the OH group of the sphingosine moiety and the phosphate ester oxygen of the head group. The narrower linewidth and the downfield shift of the resonance corresponding to OH proton in SM suggest that this H-bond is stronger in SM than in DHSM. The NH group appears to be involved predominantly in intramolecular H-bonding in the monomer. As the concentration of SM increases and the molecules come in closer proximity, these intramolecular bonds are partially disrupted and the NH group becomes involved in lipid-water interactions. The difference between the SM and DHSM appears to be not in the nature of these interactions but rather in the degree to which these intermolecular interactions prevail. As SM molecules cannot come as close together as DHSM molecules can, both the NH and OH moieties remain, on average, more intramolecularly bonded as compared to DHSM.

A 'fail-safe' screening programme for diabetic retinopathy.

OBJECTIVE: To improve screening for diabetic retinopathy in a hospital diabetic clinic through the use of the audit process. DESIGN: Comparison of an existing system of screening for diabetic retinopathy (a specialist optometrist using ophthalmoscopy alone) with a new system in which a specialist optometrist examined retinal Polaroid photographs taken through pharmacologically dilated pupils and combined this with ophthalmoscopy in all cases except when the photographs were perfect and definitely showed no retinopathy. In this new system, the optometrist could discuss cases of uncertainty with a diabetes physician while the patient was still in the clinic with eyes dilated. SETTING: Inner city hospital diabetes clinic. SUBJECTS: 289 hospital diabetic clinic patients not already attending an ophthalmologist; a consecutive series of 144 such patients for the first audit, 145 for the repeat audit. MAIN OUTCOME MEASURES: Assessment of each screening system against a gold standard. For the first audit this was agreement by two of four diabetes physicians, who combined examination of the photographs with the findings from dilated ophthalmoscopy, on the classification of the retinae of each patient, guided by standard European criteria. For the second audit, the gold standard was enhanced by discussing the photographs and findings of all patients with an independent ophthalmologist. For patients requiring referral, a second ophthalmologist also commented on the case. RESULTS: The addition of retinal photography to universal pupil dilatation, and the availability of diabetes physician backup to discuss cases of uncertainty, greatly increased the optometrists' detection rate. Sensitivities for the first (ophthalmoscopy only) and second (ophthalmoscopy plus photography plus diabetologist back-up) audits were, respectively, 71.4% vs 100% for sight-threatening retinopathy, 33% vs 100% for retinopathy requiring six-month review, and 40.3% vs 97.2% for any retinopathy (p = 0.002). CONCLUSIONS: Optometrists specialising in diabetic retinopathy using Polaroid retinal photography and ophthalmoscopy, both through dilated pupils, backed up by experienced diabetologists to discuss cases of uncertainty, could form the basis of a retinopathy screening service that accurately identifies and categorises retinopathy and does not miss sight-threatening cases.

Neural and glial-mediated effects of growth factors acting via tyrosine kinase receptors on luteinizing hormone-releasing hormone neurons.

It is becoming increasingly evident that the secretory activity of LHRH neurons is regulated not only by transsynaptic inputs but also by trophic molecules of glial and neuronal origin. The present experiments were undertaken to gain insights into the potential cell-cell mechanisms by which basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha), two growth factors produced in the hypothalamus, may affect LHRH neuronal function. Northern blot analysis showed that the LHRH-producing cell line GT1-7 contains the messenger RNA (mRNA) encoding the type 1 fibroblast growth factor receptor (FGFR-1) but not that encoding the epidermal growth factor (EGF) receptors, which mediates the biological actions of both TGF alpha and EGF. Ligand-induced receptor phosphorylation experiments demonstrated that GT1-7 cells possess biologically active FGFR-1s but not EGF receptors. Exposure of the cells to bFGF resulted not only in FGFR-1 tyrosine phosphorylation, but also in tyrosine phosphorylation of phospholipase C gamma, one of the initial enzymes in the intracellular signaling cascade initiated by FGFR activation. GT1-7 cells proliferated in response to this activation. Despite the presence of biologically active receptors, bFGF did not significantly stimulate release of the mature LHRH decapeptide. Instead, bFGF increased the steady-state levels of the mRNA encoding the LHRH precursor processing endoprotease PC2, with a time course comparable to that of phorbol esters, suggesting that, as shown in the companion paper, the actions of the growth factor on LHRH neurons involve facilitation of the initial step in LHRH prohormone processing. The increase in PC2 gene expression was not accompanied by changes in LHRH mRNA levels. Unlike these direct actions of bFGF on GT-1 cells, TGF alpha appears to act indirectly via astroglial intermediacy. Exposure of GT1-7 cells to TGF alpha or EGF failed to affect several parameters of cellular activity including LHRH release, LHRH and PC2 mRNA levels, and cell proliferation. In contrast, astrocyte culture medium conditioned by treatment with TGF alpha led to sustained stimulation of LHRH release with no changes in LHRH gene expression and a transient increase in PC2 mRNA levels. Although no definitive evidence for the presence of FGFR-1 in normal LHRH neurons could be obtained by either double immunohistochemistry or double in situ hybridization procedures, fetal LHRH neurons in primary culture responded to bFGF with neurite outgrowth. Thus, normal LHRH neurons may have an FGFR-1 content too low for detection by regular histochemical procedures, and/or detectable expression of the receptor may be confined to a much earlier developmental stage. The mitogenic effect of bFGF on GT1-7 cells supports this possibility and suggests a role for FGF in the cell proliferation events that precede acquisition of the LHRH neuronal phenotype. It appears that once this phenotype is established, bFGF may promote the differentiation of LHRH neurons. The results also suggest that the secretory capacity of LHRH neurons develops under a dual trophic influence, one on peptide processing exerted directly by bFGF on early neurons, and another on LHRH release, exerted by TGF alpha via the intermediacy of astroglial cells.

Multiple glucan-binding proteins of Streptococcus sobrinus.

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.

Streptococcal glucan-binding lectins do not recognize methylated alpha-1,6 glucans.

The glucan-binding lectin (GBL) of Streptococcus sobrinus is cell associated, enabling the bacteria to be aggregated by alpha-1,6 glucans. Glucans, such as amylose, pullulan, laminarin and nigeran, have no affinity for the lectin. High molecular weight alpha-1,6 glucans (dextrans) readily aggregate the bacteria, whereas low molecular weight glucans inhibit the aggregation brought about by the high molecular weight species. Methylated glucan T-2000 (an alpha-1,6 glucan with an average molecular weight of 2 x 10(6) Da) aggregated the bacteria very poorly when the extent of methylation (DS, or degree of substitution) was high, and less poorly when the DS was low. Similarly, methylated low molecular weight alpha-1,6 glucan was a poor inhibitor of aggregation induced by the high molecular weight glucan T-2000. Because the methylation occurred primarily on the hydroxyl of C-2, it is suggested that the hydroxyl is needed for formation of the lectin-glucan complex. It appears that the GBL is not only stereospecific in interaction with glucans, but also regio-specific, interacting only with the underivatized alpha-1,6-glucan.

Subinhibitory concentrations of antibiotics affect cell surface properties of Streptococcus sobrinus.

Several antibiotics, at subinhibitory concentrations, caused an increase in the ability of Streptococcus sobrinus to bind alpha-1,6-glucans, whereas other antibiotics decreased glucan binding. In every case, glucan binding was inversely proportional to cell surface hydrophobicity. High levels of glucan-binding activity resulted in low levels of hydrophobicity, whereas low levels of glucan binding caused high levels of cellular hydrophobicity. The results show that low concentrations of antibiotics may modulate lectin and hydrophobin adhesins in streptococci.

Separation and characterization of the unknown phospholipid in human lens membranes.

PURPOSE: The major component of human lens membranes was thought to be sphingomyelin until 1991, when a study by phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy revealed the presence of an unknown phospholipid that constituted approximately half the human lens phospholipids. The objective of this work was to isolate this phospholipid and to elucidate its identity. METHODS: The separation of sphingomyelin from the unknown was accomplished using high-performance liquid chromatography (HPLC) and an amino-bound column. Sphingomyelin standard and the membranes from human lenses were chromatographed. Chromatographic fractions were collected and spectrally characterized by proton (1H) NMR and 31P NMR spectroscopy. RESULTS: The chromatographic method did not affect the integrity of the sphingomyelin. Besides the bands corresponding to the unknown components, the chromatogram of the human lens membranes showed three large peaks, the central one with a shoulder, with elution times similar to that for sphingomyelin. The 1H NMR spectra for the fractions collected during the elution of these peaks showed differences. The study by 31P NMR indicated that the first peak contained the unknown phospholipid. The subsequent fractions showed the presence, in different relative levels, of both the unknown and sphingomyelin. By comparison and interpretation of the two-dimensional 1H NMR spectra for sphingomyelin and for the fraction containing the unknown, the unknown phospholipid is proposed to be 4,5 dihydrosphingomyelin, in which the site of unsaturation present in the sphingosine moiety is no longer present. CONCLUSIONS: The ability to separate the unknown from sphingomyelin and the power of 1H NMR spectroscopy allowed the proposition of the identity of the major component of human lens membranes as 4,5-dihydrosphingomyelin. Although the synthetic compound is known to be involved in the formation of extended hydrogen-bonding networks, its biologic and physicochemical properties need further study.

Fluoride inhibits the glucan-binding lectin of Streptococcus sobrinus.

The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.

Hyperthyroidism associated with hyperemesis gravidarum.

Hyperemesis gravidarum is an uncommon presentation of hyperthyroidism in pregnancy which is usually attributable to autoimmune (Graves') disease. While this condition necessitates treatment with antithyroid drugs, a syndrome of transient hyperthyroidism associated with hyperemesis gravidarum that resolves spontaneously is also recognised. Differentiation between these two conditions may prove problematic in practice. We report two cases of hyperthyroidism associated with severe hyperemesis gravidarum. Intractable hyperemesis continued in one patient despite normalisation of circulating free thyroid hormone concentrations with carbimazole. Neither patient exhibited clinical or immunological features of autoimmune thyroid disease, suggesting in retrospect that they had the syndrome of transient hyperthyroxinaemia associated with hyperemesis gravidarum rather than Graves' disease. The role of antithyroid drugs in the treatment of self-limiting transient hyperthyroidism associated with hyperemesis gravidarum requires clarification.

ACTH-independent Cushing's syndrome in pregnancy with spontaneous resolution after delivery: control of the hypercortisolism with metyrapone.

A 25-year-old primigravid woman presented with Cushing's syndrome at 23 weeks gestation; serum cortisol was 1090 nmol/l at 0900 h, 1230 nmol/l at 2200 h; basal urinary free cortisol excretion was 3680 nmol/24 h, and 8830 nmol/24 h after dexamethasone 8 mg daily for 48 hours; plasma ACTH was < 1.1 pmol/l. CT scan of the adrenal glands showed bilateral adrenal hyperplasia. The hypercortisolism was controlled with metyrapone until elective delivery of the fetus by Caesarean section at 34 weeks gestation because of a decline in growth. No adverse fetal effects of metyrapone treatment were apparent, maternal outcome was uncomplicated and wound healing was unimpaired. Maternal adrenocortical function had returned to normal within 4 weeks of the cessation of pregnancy and biochemical remission has been maintained up to 9 months post-partum. Metyrapone therapy is effective in controlling the hypercortisolism in certain cases of Cushing's syndrome complicating pregnancy.

Diagnosis of Pneumocystis carinii infection in HIV-seropositive patients by identification of P carinii in pleural fluid.

Pneumocystis carinii pneumonia (PCP) is the most common pulmonary complication of AIDS and is typically diagnosed by the identification of P carinii organisms in sputum, bronchoalveolar lavage fluid, or tissue obtained with transbronchial biopsy. We describe two HIV-seropositive patients with pleural effusions in whom the diagnosis of P carinii infection was made by examination of pleural fluid. Pleural effusions associated with PCP are very unusual but can provide a source of diagnostic material particularly in those HIV patients who have development of a spontaneous pneumothorax and require chest tube insertion.

Essential amino acids involved in glucan-dependent aggregation of Streptococcus sobrinus.

The active site of the glucan-binding lectin (or agglutinin) (GBL) of Streptococcus sobrinus was probed by specific amino acid modifying reagents. Reagents specific for carboxylates, imidazolium, phenolic, and lysyl residues inactivated the cell bound GBL, whereas agents specific for sulfhydryl, disulfide, and guanidinium groups had no effect on the lectin. A low molecular weight alpha-(1-->6)-glucan provided partial protection against the reagents which inactivated the protein, whereas an alpha-(1-->4)-glucan, incapable of complexing with the lectin, afforded no protection. A reagent specific for tryptophan, 2-hydroxy-5-nitrobenzyl bromide (HNB) did not cause a loss of GBL activity, although N-bromosuccinimide, a reagent capable of oxidizing tryptophan and less selective than HNB, was a very effective inhibitor of the glucan-dependent cellular aggregation. In the latter case, alpha-(1-->6)-glucan did not protect. Hydroxylamine partially restored the loss of lectin activity due to treatment of the cells with N-acetylimidazole (highly specific for tyrosine), glycine methyl ester plus water-soluble carbodiimide (specific for carboxylates), and diethylpyrocarbonate (specific for histidine). Because the soluble form of GBL rapidly loses activity when purified, it was necessary to perform the chemical modification of the amino acid side chains employing the cell-bound form of the lectin. Because specific ligand [alpha-(1-->6)-glucan] protected against the inactivation of the agglutinin by selected reagents and because lectin activity could be restored in some cases, it was possible to identify likely essential amino acid residues needed for glucan binding. The results, taken together, suggest that aspartic (and/or glutamic) acid, histidine, lysine, and tyrosine are critical amino acids responsible for agglutinin activity. Present efforts are directed to the design and synthesis of glucan analogues which may serve as affinity inactivating agents of the lectin. Such glucan derivatives may be of value in studies on the role of the lectin in cariogenesis.