Neither Moderate Hypoxia nor Mild Hypoglycaemia Alone Causes Any Significant Increase in Cerebral [Ca2±i: Only a Combination of the Two Insults Has This Effect.: A 31P and 19F NMR Study

(1) The energy state and free intracellular calcium concentration ([Ca^2±_i) of super-fused cortical slices were measured in moderate hypoxia (～65 μM O₂), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using ¹⁹F and ³¹P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca^2±_i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca^2±, and a decrease in PCr level to ～25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca^2±_i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca^2±_i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca^2±_i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.     

Human papillomavirus type 16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation.

The transforming function of human papillomavirus type 16 (HPV16) E7 has been shown to depend on activities additional to the ability to bind RB. In this paper we describe two further properties of E7 which may also contribute to transformation, an association with a histone H1 kinase at the G2/M phase of the cell cycle and an ability to bind the RB-related protein p107. The region of E7 identified previously as important for RB binding was found to be involved in the association with the kinase and complex formation with p107, although analysis of E7 point mutants within this region revealed a difference in the precise sequence requirement for RB and p107 binding. Association with the kinase activity correlated with the ability to bind RB, but the restriction of the kinase association to the G2/M phase of the cell cycle implies that this activity might not be directly mediated by RB binding. Since kinase-binding-deficient E7 mutants are also transformation defective, this may represent an independent function of E7 which plays a role in the G2/M phase of the cell cycle.     

Chromosome 14 and late-onset familial Alzheimer disease (FAD)

Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analyses were used, in 49 families with a mean age at onset ≥60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependent penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score ≤ –2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P < .02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52, however, was found for a subgroup of families of intermediate age at onset (mean age at onset ≥60 years and <70 years). These results indicate that the chromosome 14 locus is not responsible for Alzheimer disease in most late-onset FAD kindreds but could play a role in a subset of these kindreds.     

Either alpha-tubulin isogene product is sufficient for microtubule function during all stages of growth and differentiation in Aspergillus nidulans.

The filamentous fungus Aspergillus nidulans has two genes encoding alpha-tubulin, tubA and tubB, which are differentially required at distinct stages during the life cycle. The tubA gene is required during vegetative growth for mitosis and nuclear migration (B. R. Oakley, C. E. Oakley, and J. E. Rinehart, Mol. Gen. Genet. 208:135-144, 1987; P. Doshi, C. A. Bossie, J. H. Doonan, G. S. May, and N. R. Morris, Mol. Gen. Genet. 225:129-141, 1991). The tubB gene is not required for any detectable aspect of vegetative growth or asexual reproduction but is essential during sexual development prior to the first meiotic division (K. E. Kirk and N. R. Morris, Genes Dev. 5:2014-2023, 1991). In this study, we determined whether the role of each alpha-tubulin gene is to provide a specific isotype necessary for a particular microtubule function or whether either alpha-tubulin isotype, if present in sufficient quantities, can participate effectively in all types of microtubule. Strains carrying a deletion allele of tubB (tubB delta) produce no ascospores from a cross. When one copy of a plasmid containing the region upstream of the tubB gene fused to the tubA coding region was integrated into a tubB delta strain, ascosporogenesis proceeded beyond the tubB delta block and resulted in the formation of sexual spores. However, irregular numbers of spores formed in some asci during development, and the ascospores had greatly diminished viability and aberrant morphologies. These defects were nearly corrected when two additional copies of the tubA coding region were integrated into the tubB delta strain. These results indicate that the tubA alpha-tubulin isotype can form functional microtubules during sexual development in the absence of tubB protein. In a reciprocal set of experiments, we examined whether upregulation of tubB can complement the tubA4 mutation, which causes supersensitivity to benomyl during vegetative growth. When tubA4 strains integrated a plasmid containing an alcohol-inducible promoter joined to the tubB coding region and subsequently overexpressed the tubB isotype, the benomyl supersensitivity normally caused by the tubA4 allele was relieved. These results indicate that when enough tubB alpha-tubulin is supplied, strains lacking functional tubA isotype can still form microtubules which effectively carry out mitosis and nuclear migration.     

Causal inference and large-scale expert validation shed light on the drivers of SDM accuracy and variance

Aim

To develop a causal understanding of the drivers of Species distribution model (SDM) performance.

Location

United Kingdom (UK).

Methods

We measured the accuracy and variance of SDMs fitted for 518 species of invertebrate and plant in the UK. Our measure of variance reflects variation among replicate model fits, and taxon experts assessed model accuracy. Using directed acyclic graphs, we developed a causal model depicting plausible effects of explanatory variables (e.g. species' prevalence, sample size) on SDM accuracy and variance and quantified those effects using a multilevel piecewise path model.

Results

According to our model, sample size and niche completeness (proportion of a species' niche covered by sampling) directly affect SDM accuracy and variance. Prevalence and range completeness have indirect effects mediated by sample size. Challenging conventional wisdom, we found that the effect of prevalence on SDM accuracy is positive. This reflects the facts that sample size has a positive effect on accuracy and larger sample sizes are possible for widespread species. It is possible, however, that the omission of an unobserved confounder biased this effect. Previous studies, which reported negative correlations between prevalence and SDM accuracy, conditioned on sample size.

Main conclusions

Our model explicates the causal basis of previously reported correlations between SDM performance and species/data characteristics. It also suggests that niche completeness has similarly large effects on SDM accuracy and variance as sample size. Analysts should consider niche completeness, or proxies thereof, in addition to sample size when deciding whether modelling is worthwhile.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Abnormalities of sleep in patients with the chronic fatigue syndrome.

OBJECTIVE--To determine whether patients with the chronic fatigue syndrome have abnormalities of sleep which may contribute to daytime fatigue. DESIGN--A case-control study of the sleep of patients with the chronic fatigue syndrome and that of healthy volunteers. SETTING--An infectious disease outpatient clinic and subjects'' homes. SUBJECTS--12 patients who met research criteria for the chronic fatigue syndrome but not for major depressive disorder and 12 healthy controls matched for age, sex, and weight. MAIN OUTCOME MEASURES--Subjective reports of sleep from patients'' diaries and measurement of sleep patterns by polysomnography. Subjects'' anxiety, depression, and functional impairment were assessed by interview. RESULTS--Patients with the chronic fatigue syndrome spent more time in bed than controls (544 min v 465 min, p < 0.001) but slept less efficiently (90% v 96%, p < 0.05) and spent more time awake after initially going to sleep (31.9 min v 16.6 min, p < 0.05). Seven patients with the chronic fatigue syndrome had a sleep disorder (four had difficulty maintaining sleep, one had difficulty getting to sleep, one had difficulty in both initiating and maintaining sleep, and one had hypersomnia) compared with none of the controls (p = 0.003). Those with sleep disorders showed greater functional impairment than the remaining five patients (score on general health survey 50.4% v 70.4%, p < 0.05), but their psychiatric scores were not significantly different. CONCLUSIONS--Most patients with the chronic fatigue syndrome had sleep disorders, which are likely to contribute to daytime fatigue. Sleep disorders may be important in the aetiology of the syndrome.     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

ELECTRON MICROSCOPY OF OSTEOCLASTS IN HEALING FRACTURES OF RAT BONE

Osmium-fixed, undecalcified, callus tissue from healing fractures of rat tibias was sectioned with a diamond knife for study with the electron microscope. Large multinucleated cells were found adjacent to bone. A characteristic labyrinthine infolded border was consistently seen in parts of the cells close to the bone surface. The innermost parts of this "ruffled border" gave rise to vacuoles. The bone surface was always disrupted under the "ruffled border" of the cells. Needle-like crystals were seen at the osseous fringe, within folds in the ruffled border as well as within vacuoles deeper in the cells. Collagen fibers denuded of crystals were never observed. Mitochondria, containing clusters of fine granules, were abundant. The part of the cell away from bone contained rough endoplasmic reticulum and the cell membrane was thrown into irregular microvilli. These observations are discussed in relation to current concepts of osteoclastic resorption of bone.