Inflammation and neurovascular changes in amyotrophic lateral sclerosis.

Neuroinflammation in now established as an important factor in the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). At various time points, astrocytes and microglia are markedly activated, either producing neuroprotective or pro-inflammatory molecules, which can decrease or increase the rate of primary motor neuron degeneration respectively. Recent research has shown that this neuroinflammatory component is affected by the peripheral immune system; T lymphocytes in particular are able to cross into the brain and spinal cord parenchyma, where they interact with resident microglia, either inducing them to adopt an M1 (cytotoxic) or M2 (protective) phenotype, depending on the stage of disease. Clearly understanding the changes that occur to allow the interaction between peripheral and central immune responses will be essential in any attempt to manipulate the disease process via neuroinflammatory mechanisms. However, our understanding of the endothelial changes, which facilitate the infiltration of peripheral immune cells into the brain and spinal cord, is still in its infancy. There are suggestions, though, of up-regulation of cellular adhesion molecules, which are able to arrest circulating leukocytes and facilitate diapedesis into the brain parenchyma. In addition, tight junction proteins appear to be down-regulated, leading to an increase in vascular permeability, an effect that is amplified by vascular damage late in the disease process. This review summarises our current knowledge regarding neuroinflammation, peripheral immune involvement, and endothelial changes in ALS. This article is part of a Special Issue entitled 'Neuroinflammation in neurodegeneration and neurodysfunction'.

A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism Streptomyces coelicolor A3(2).

A simple and high-throughput transposon mediated mutagenesis system employing in vitro shuttle transposon mutagenesis has been used to systematically mutagenise the Streptomyces coelicolor genome. To achieve the highest coverage, a new ordered cosmid library was also constructed. Individual cosmids from both the existing and new libraries were disrupted using the Tn5-based mini-transposon Tn5062. A total of 35,358 insertions were sequenced resulting in the disruption of 6,482 genes (83% of the predicted open reading frames). Complete information for both the newly generated cosmids as well as all the insertions has been uploaded onto a central database, StrepDB ( http://strepdb.streptomyces.org.uk/ ). All insertions, new cosmids and a range of transposon exchange cassettes are available for study of individual gene function.

Novel embryonic genes are preferentially expressed by autonomously replicating rat embryonic and neointimal smooth muscle cells.

We sought to identify and characterize the expression pattern of genes expressed by smooth muscle cells (SMCs) during periods of self-driven replication during vascular development and after vascular injury. Primary screening of a rat embryonic aortic SMC-specific cDNA library was accomplished with an autonomous embryonic SMC-enriched, nonautonomous adult SMC-subtracted cDNA probe. Positive clones were rescreened in parallel with embryonic SMC-specific and adult SMC-specific cDNA probes. We identified 14 clones that hybridized only with the embryonic cDNA ("emb" clones), 11 of which did not share significant homology with sequences in any of the databases. Five of these novel emb genes (emb7, emb8, emb20, emb37, and emb41) were selectively and only transiently reexpressed in vivo by neointimal SMCs during periods of rapid replication. The emb8:embryonic growth-associated protein (EGAP), which was studied the most extensively, was expressed at high levels by cultured, autonomously replicating embryonic and neointimal SMCs but was detected only at low levels even in mitogenically stimulated adult SMCs. Finally, the administration of antisense EGAP oligonucleotides markedly attenuated embryonic and neointimal SMC replication rates. We suggest that autonomous replication of SMCs may be essential for normal vascular morphogenesis and for the vascular response to injury and that these newly identified "embryonic" genes may be part of the molecular machinery that drives this unique growth phenotype.

Investigation of the ammonium chloride and ammonium acetate inhibition of oxygen evolution by Photosystem II.

Using EPR and EXAFS spectroscopies we show that high concentrations of ammonium cations at alkaline pH are required for (1) inhibition of oxygen evolution: (2) an alteration of the EPR properties of the oxygen evolving complex: (3) the ability to detect YZ; and (4) the slow reduction of the Mn complex leading to the appearance of EPR detectable Mn2+. The inhibition of S state cycling, slowing of YZ reduction, appearance of Mn2+ and the yield of a Hpp < 10 mT S3 type EPR signal are decreased by calcium addition. This indicates that these effects were probably associated with calcium depletion arising from the high concentration of ammonium cation. The ammonia-induced changes to the S2 multiline EPR signal are not affected by calcium addition. The appearance of Mn2+ is shown to be reversible on illumination, suggesting that the Mn reduced from the native state is located at or near the native site. Simulations of the interaction which give rise to the S3 EPR signal are also presented and discussed. These indicate that lineshape differences occur through small changes in the exchange component of the interaction between the manganese complex and organic radical, probably through minor structural changes between the variously treated samples.

Photosynthetic water oxidation: towards a mechanism.

This mini-review outlines the current theories on the mechanism of electron transfer from water to P680, the location and structure of the water oxidising complex and the role of the manganese cluster. We discuss how our data fit in with current theories and put forward our ideas on the location and mechanism of water oxidation.

Comparison of the EPR properties of photosystem I iron-sulphur centres A and B in spinach and barley.

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.

EPR characteristics of oxygen-evolving photosytem II particles from Phormidium laminosum.

The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome beta-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described. The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated alga membranes for the study of Photosystem II with a functional water oxidation system.

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth.

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.

The role of the membrane-bound iron-sulphur centres A and B in the photosystem I reaction centre of spinach chloroplasts.

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I (P-700) and X. Oxidation-reduction potential titrations confirmed that Centre A had Em congruent to -550 mV, Centre B had Em congruent to -585 mV. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, P-700 photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible P-700 photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with Em congruent to -585 mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.

The two forms of the S(2) state multiline signal in Photosystem II: effect of methanol and ethanol.

The characteristic Mn hyperfine 'multiline' signal exhibited in the S2 state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S2 state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol.

Testosterone therapy in glucocorticoid-treated men.

BACKGROUND: Treatment with glucocorticoid drugs is a valuable therapy, but the use of these drugs is associated with major side effects, including osteoporosis, muscle wasting, and obesity. In men who take glucocorticoids, circulating testosterone concentrations are reduced, and this might contribute to the changes in bone and soft-tissue mass. OBJECTIVE: To asses the effect of testosterone replacement on these above-mentioned parameters in glucocorticoid-treated men. METHODS: Fifteen asthmatic men who were receiving long-term glucocorticoid treatment were randomly allocated to receive therapy with testosterone esters (30 mg of proprionate, 60 mg of phenylprionate, 60 mg of isocaproate, and 100 mg of decanoate [Sustanon]) (250-mg/mo intramuscular depot injection) or to act as control subjects during 12 months. After a washout period for those men who were receiving testosterone, the groups were then crossed over and studied for a further 12 months. Bone density and body composition were assessed by dual-energy, x-ray absorptiometry. Paired or unpaired 2-tailed t tested were calculated. Unless otherwise stated, all values are given as mean +/- SEM. RESULTS: Bone density in the lumbar spine increased 5.0% +/- 1.4% (mean +/- SEM) (P = .005) during testosterone supplementation, but it did not change during the control period (between-groups difference, P = .05). These changes were accompanied by a decrease in the indexes of bone turnover. There was a gain in body fat mass (2.1 +/- 0.06 kg, P = .01) and a loss of lean body mass (1.4 +/- 0.5 kg, P = .02) during the control period, with both changes being reversed by testosterone treatment (P < .03). CONCLUSION: Testosterone treatment reverses the deleterious effects glucocorticoid drugs on skeletal and soft tissues in men.

Effects of hormone replacement therapy on bone mineral density in postmenopausal women with primary hyperparathyroidism: four-year follow-up and comparison with healthy postmenopausal women.

BACKGROUND: Long-term treatment of patients with asymptomatic primary hyperparathyroidism remains controversial, but the presence of osteoporosis is regarded as an indication for parathyroidectomy. Hormone replacement therapy (HRT) is a possible alternative therapy in osteopenic postmenopausal women with the disorder, and results of short-term studies suggest a beneficial effect on bone mass comparable to that achieved by parathyroidectomy. Longer-term data are required to further assess the efficacy of this treatment in chronic stable primary hyperparathyroidism. METHODS: We report the results of the extension from 2 to 4 years of a randomized, placebo-controlled trial of HRT in postmenopausal women with primary hyperparathyroidism. Of 23 postmenopausal women with primary hyperparathyroidism, 11 received active HRT with conjugated equine estrogen, 0.625 mg/d, and medroxyprogesterone acetate, 5 mg/d, and 12 received placebo. Bone mineral density was measured throughout the skeleton at 6-month intervals using dual-energy x-ray absorptiometry in these women and in 50 normocalcemic age-matched control subjects. None of the 23 patients withdrew during the extension period. RESULTS: Changes in bone mineral density were more positive in those taking HRT than placebo, with the between-group differences at 4 years being 4.6% in the total body, 7.5% in the lumbar spine, 7.4% in the femoral neck, 8.2% in the femoral trochanter, 6.8% in the legs, and 7.0% in the forearm (P<.01). At skeletal sites composed predominantly of cortical bone, there was a progressive divergence of the 2 groups. Biochemical markers of bone turnover remained lower throughout the study in women taking HRT. When rates of bone loss were compared between the placebo group and healthy women of comparable age, bone loss tended to be more marked throughout the skeleton in women with hyperparathyroidism, but only in the total body and its legs subregion was this difference significant. CONCLUSIONS: Hormone replacement therapy is efficacious in the long-term management of osteopenia in postmenopausal women with primary hyperparathyroidism and thus represents an important new therapeutic option for asymptomatic patients who do not have other indications for surgery. Bone loss seems to be accelerated in untreated primary hyperparathyroidism.

Insulin Does Not Target CamkIIα Neurones to Critically Regulate the Neuroendocrine Reproductive Axis in Mice.

Insulin signalling in the brain plays an important role in the central regulation of energy homeostasis and fertility, such that mice exhibiting widespread deletion of insulin receptors (InsR) throughout the brain and peripheral nervous system display diet sensitive obesity and hypothalamic hypogonadism. However, the specific cell types mediating the central effects of insulin on fertility remain largely unidentified. To date, the targeted deletion of InsR from individual neuronal populations implicated in the metabolic control of fertility has failed to recapitulate the hypogonadic and subfertile phenotype observed in brain-specific InsR knockout mice. Because insulin and leptin share similar roles as centrally-acting metabolic regulators of fertility, we used the Cre-loxP system to generate mice with a selective inactivation of the Insr gene from the same widespread neuronal population previously shown to mediate the central effects of leptin on fertility by crossing Insr-flox mice with calcium/calmodulin-dependent protein kinase type IIα (CamkIIα)-Cre mice. Multiple reproductive and metabolic parameters were then compared between male and female Insr-flox/Cre-positive (CamK-IRKO) and Insr-flox/Cre-negative control mice. Consistent with brain-specific InsR knockout mice, CamK-IRKO mice exhibited a mild but significant obesogenic phenotype. Unexpectedly, CamK-IRKO mice exhibited normal reproductive maturation and function compared to controls. No differences in the age of puberty onset, oestrous cyclicity or fecundity were observed between CamK-IRKO and control mice. We conclude that the central effects of insulin on the neuroendocrine reproductive axis are not critically mediated via the same neuronal populations targeted by leptin.

Lateral spine densitometry is a more sensitive indicator of glucocorticoid-induced bone loss.

Osteoporosis is a common complication of glucocorticoid therapy. Bone density measurement is now commonly used in assessing which steroid-treated patients require specific interventions to reduce fracture risk. The recently developed techniques for the measurement of bone mineral density (BMD) of the vertebral body alone, by dual-energy x-ray absorptiometry (DXA) in the lateral projection, may be particularly useful in this context since steroid-induced bone loss is most marked in trabecular-rich regions like the vertebral body. This possibility has been assessed in the present study by the measurement of BMD in the lateral and anterioposterior (AP) projections in 28 women receiving chronic glucocorticoid treatment. The two BMD measurements were significantly related (r = 0.62, p < 0.001). When expressed in relation to age-appropriate normal values, lateral BMDs were lower than AP BMDs both in percentage terms (70.8 +/- 4.4 versus 90.3 +/- 2.6%, p < 0.001) and in terms of Z scores (-1.42 +/- 0.22 versus -0.91 +/- 0.24, p = 0.027). AP BMD Z scores classified 12 patients as osteopenic, whereas a further 7 were so categorized by lateral BMD Z score. It is concluded that lateral DXA scanning is a more sensitive indicator of glucocorticoid-induced osteopenia than conventional BMD measurement in the AP projection.

Sources of interracial variation in bone mineral density.

Many studies have demonstrated significant differences in bone mineral density between various racial groups. Although it has been suggested that differences in body weight contribute to such interracial variation, the artifactual effect of the skeletal size inherent in projectional absorptiometry methods has been largely ignored. We have measured bone mineral density by dual-energy X-ray absorptiometry in the lumbar spine and at three femoral sites in 200 premenopausal women of Chinese, Indian, European, or Polynesian origin (50 of similar mean age in each group). In the Chinese and Indian women the measured bone mineral density measurements (g/cm2) were similar, but significantly less, at all sites, than those of European women (p < or = 0.005). The European women were, however, significantly taller than both the Chinese and Indian women (p < 0.0001), and when the scale artifact of absorptiometry was removed by dividing the measured bone mineral density either by the height of the subject, or by the square root of the area over which the X-ray beam was projected, then the differences in mean bone mineral density between the Chinese, Indian, and European women were almost completely eliminated. The Polynesian women were significantly more obese (as judged from mean body mass index) than all the other groups (p < 0.0001) and had significantly greater bone mineral density at all sites than all the other groups both before (p < 0.0001) and after (p < 0.0001) correcting for the scale artifact.(ABSTRACT TRUNCATED AT 250 WORDS)

Fat mass is an important determinant of whole body bone density in premenopausal women but not in men.

We recently reported that total body fat mass is the principal determinant of bone density in normal postmenopausal women. We have now reexamined the relationships among these variables and lean mass in 68 healthy premenopausal women and 51 men. Areal bone density (BMD), fat mass, and lean mass were measured in total body scans by dual-energy, x-ray absorptiometry. In women, BMD was correlated with weight (r = 0.69), fat mass (r = 0.60), and lean mass (r = 0.55). In men, the respective correlations were 0.56, 0.26 (NS), and 0.51. Multiple regression analysis confirmed a codependence of female BMD on fat and lean masses, whereas male BMD was related only to lean mass. Because BMD is an areal not volumetric density, it is dependent on body size. The analysis was therefore repeated using BMD/height as an index of "true" density. Correlations with fat mass were little changed but those with lean mass were reduced (women) or eliminated (men). By multiple regression, female BMD/height was related to fat mass alone, and in men there was a borderline effect of fat (P = 0.05) but none of lean mass. As a second method to exclude a scale artifact, fat mass was expressed as percent body weight. It was related to BMD (r = 0.48) only in women. It is concluded that bone density is closely related to fat mass in premenopausal women, but less so in men. In both sexes, apparent relationships between BMD and lean mass are artifacts attributable to the use of areal density (which is dependent on body size) as a surrogate for volumetric density. The mechanism of this fat-bone density relationship is an important question to be addressed in bone biology.

Growth hormone-like material in normal human tissues.

We report herein the presence of extractable levels of immunoassayable (RIA) and receptor-assayable human GH (hGH)-like material in normal human liver, kidney, lung, striated muscle, colon, stomach, and brain. Levels of hGH by RIA were the highest in the liver (mean, 14.7 ng/g), followed by those in the kidney (mean, 11.3 ng/g) and lung (mean, 4.6 ng/g), but small amounts were detected in at least some specimens of all types of tissue. This material eluted in the region of monomeric pituitary hGH on Sephadex G-100 chromatography and showed parallelism in a RIA to pituitary hGH on log-logit plots of dose-response lines. Passage of the material through an anti-hGH immunocolumn greatly reduced its immunoreactivity. Assay of eight sets of extracts by receptor assay using a male rat liver membrane preparation showed the GH-like substance to have a receptor to immunoassay potency ratio of 3.8:1 relative to monomeric hGH standard. Scrupulous correction for acidity, osmolality, and possible protein and enzyme effects and extraction of the hGH-like material by immunoabsorption excluded the possibility of artifacts causing the appearance of hGH-like material in the extracts.

The influence of osteophytes and aortic calcification on spinal mineral density in postmenopausal women.

The assessment of vertebral bone mineral density (BMD) in the anterio-posterior projection has become widely used in the management and prevention of osteoporosis. Recently, it has been demonstrated that the presence of spinal osteophytes has a major impact on measured BMD in men, thus casting doubt on the value of these BMD measurements. We have assessed the impact of osteophytic and aortic calcification on spinal and femoral BMD measurements in 130 normal postmenopausal women, aged 45-71 yr. Lateral lumbar spine radiographs were obtained in all subjects and graded separately (0-3) for osteophytes and aortic calcification. Both forms of calcification increased with age, and BMD of all sites was correlated positively with body weight and negatively with age. The correlation coefficients between BMD and calcification scores were nonsignificant. Multiple regression analysis, including weight, age, and calcification scores, demonstrated a small but significant effect of osteophyte score on lumbar BMD (partial r2 = 0.04; P = 0.012) and a similar trend for Ward's triangle and the trochanteric region (partial r2 = 0.02; P less than 0.06). The aortic calcification score remained nonsignificant. It is concluded that the influence of spinal osteophytes on lumbar BMD in postmenopausal women is substantially less than that in men and is, therefore, unlikely to interfere with BMD estimation in most subjects. The relationship between proximal femoral BMD and osteophyte score suggests a real relationship between skeletal density and degenerative joint disease, as has been demonstrated by others.

Low thyroxine levels in some hyperprolactinemic patients due to dopaminergic suppression of thyrotropin.

The serum free T4 index (FT4I) was at or below the lower limit of normal in 8 of 55 unselected patients with hyperprolactinemia. Serum levels of T3 were normal and none of the patients had clinical evidence of hypothyroidism. In patients with low FT4I the serum TSH was within the normal range and TSH was released normally after administration of TRH, indicating normal pituitary TSH reserve. Serum TSH also increased after administration of the dopamine antagonist domperidone. The TSH response to domperidone was significantly greater in the hyperprolactinemic group with low FT4I compared with either normal subjects or hyperprolactinemic patients with normal FT4I, suggesting that depression of FT4I was due to increased dopaminergic activity. Administration of the dopamine antagonist metoclopramide for 4 days led to a supranormal rise in FT4I in 3 of 5 patients with low FT4I. Thus, a minority of hyperprolactinemic patients have a low FT4I which appears due to excessive hypothalamic production of dopamine.