Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analysed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis – dynasore and tat-gluR2(3Y) – were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process. (220)

Prenatal inhibition of the tryptophan–kynurenine pathway alters synaptic plasticity and protein expression in the rat hippocampus

Glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) are important in early brain development, influencing cell proliferation and migration, neuritogenesis, axon guidance and synapse formation. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Rats were treated in late gestation with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]-benzene-sulphonamide (Ro61-8048), an inhibitor of kynurenine-3-monoxygenase which diverts kynurenine metabolism to kynurenic acid. Within 5 h of drug administration, there was a significant decrease in GluN2A expression and increased GluN2B in the embryo brains, with changes in sonic hedgehog at 24 h. When injected dams were allowed to litter normally, the brains of offspring were removed at postnatal day 21 (P21). Recordings of hippocampal field excitatory synaptic potentials (fEPSPs) showed that prenatal exposure to Ro61-8048 increased neuronal excitability and paired-pulse facilitation. Long-term potentiation was also increased, with no change in long-term depression. At this time, levels of GluN2A, GluN2B and postsynaptic density protein PSD-95 were all increased. Among several neurodevelopmental proteins, the expression of sonic hedgehog was increased, but DISC1 and dependence receptors were unaffected, while raised levels of doublecortin and Proliferating Cell Nuclear Antigen (PCNA) suggested increased neurogenesis. The results reveal that inhibiting the kynurenine pathway in utero leads to molecular and functional synaptic changes in the embryos and offspring, indicating that the pathway is active during gestation and plays a significant role in the normal early development of the embryonic and neonatal nervous system

Selective depletion of tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin by environmental and endogenous serine proteases: linking diet, obesity and cancer

Background: The related tumour suppressor proteins Deleted in Colorectal Cancer (DCC) and neogenin are absent or weakly expressed in many cancers, whereas their insertion into cells suppresses oncogenic behaviour. Serine proteases influence the initiation and progression of cancers although the mechanisms are unknown. Methods: The effects of environmental (bacterial subtilisin) and endogenous mammalian (chymotrypsin) serine proteases were examined on protein expression in fresh, normal tissue and human neuroblastoma and mammary adenocarcinoma lines. Cell proliferation and migration assays (chemoattraction and wound closure) were used to examine cell function. Cells lacking DCC were transfected with an ectopic dcc plasmid. Results: Subtilisin and chymotrypsin selectively depleted DCC and neogenin from cells at nanomolar concentrations without affecting related proteins. Cells showed reduced adherence and increased migration, but after washing they re-attached within 24 h, with recovery of protein expression. These effects are induced by chymotryptic activity as they are prevented by chymostatin and the soybean Bowman-Birk inhibitor typical of many plant protease inhibitors. Conclusions: Bacillus subtilis, which secretes subtilisin is widely present in soil, the environment and the intestinal contents, while subtilisin itself is used in meat processing, animal feed probiotics and many household cleaning agents. With chymotrypsin present in chyme, blood and tissues, these proteases may contribute to cancer development by depleting DCC and neogenin. Blocking their activity by Bowman-Birk inhibitors may explain the protective effects of a plant diet. Our findings identify a potential non-genetic contribution to cancer cell behaviour which may explain both the association of processed meats and other factors with cancer incidence and the protection afforded by plant-rich diets, with significant implications for cancer prevention

Condensation of MgS in outflows from carbon stars

The basic mechanism responsible for the widespread condensation of MgS in the outflows from carbon rich stars on the tip of the AGB is discussed with the aim of developing a condensation model that can be applied in model calculations of dust formation in stellar winds. The different possibilities how MgS may be formed in the chemical environment of outflows from carbon stars are explored by some thermochemical calculations and by a detailed analysis of the growth kinetics of grains in stellar winds. The optical properties of core-mantle grains with a MgS mantle are calculated to demonstrate that such grains reproduce the structure of the observed 30 $\mu$m feature. These considerations are complemented by model calculations of circumstellar dust shells around carbon stars. It is argued that MgS is formed via precipitation on silicon carbide grains. This formation mechanism explains some of the basic observed features of MgS condensation in dust shells around carbon stars. A weak secondary peak at about 33 ... 36 $\mu$m is shown to exist in certain cases if MgS forms a coating on SiC.Comment: 9 pages, 7 figure

On the Biological Importance of the 3-hydroxyanthranilic Acid: Anthranilic Acid Ratio

Of the major components of the kynurenine pathway for the oxidative metabolism of tryptophan, most attention has focussed on the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid, and the glutamate receptor blocker kynurenic acid. However, there is increasing evidence that the redox-active compound 3-hydroxyanthranilic acid may also have potent actions on cell function in the nervous and immune systems, and recent clinical data show marked changes in the levels of this compound, associated with changes in anthranilic acid levels, in patients with a range of neurological and other disorders including osteoporosis, chronic brain injury, Huntington’s disease, coronary heart disease, thoracic disease, stroke and depression. In most cases, there is a decrease in 3-hydroxyanthranilic acid levels and an increase in anthranilic acid levels. In this paper, we summarise the range of data obtained to date, and hypothesise that the levels of 3-hydroxyanthranilic acid or the ratio of 3-hydroxyanthranilic acid to anthranilic acid levels, may contribute to disorders with an inflammatory component, and may represent a novel marker for the assessment of inflammation and its progression. Data are presented which suggest that the ratio between these two compounds is not a simple determinant of neuronal viability. Finally, a hypothesis is presented to account for the development of the observed changes in 3-hydroxyanthranilic acid and anthranilate levels in inflammation and it is suggested that the change of the 3HAA:AA ratio, particularly in the brain, could possibly be a protective response to limit primary and secondary damage

A PET-CT study on neuroinflammation in Huntington’s disease patients participating in a randomized trial with laquinimod

Microglia activation, an indicator of central nervous system inflammation, is believed to contribute to the pathology of Huntington's disease. Laquinimod is capable of regulating microglia. By targeting the translocator protein, 11C-PBR28 PET-CT imaging can be used to assess the state of regional gliosis in vivo and explore the effects of laquinimod treatment. This study relates to the LEGATO-HD, multi-centre, double-blinded, Phase 2 clinical trial with laquinimod (US National Registration: NCT02215616). Fifteen patients of the UK LEGATO-HD cohort (mean age: 45.2 ± 7.4 years; disease duration: 5.6 ± 3.0 years) were treated with laquinimod (0.5 mg, N = 4; 1.0 mg, N = 6) or placebo (N = 5) daily. All participants had one 11C-PBR28 PET-CT and one brain MRI scan before laquinimod (or placebo) and at the end of treatment (12 months apart). PET imaging data were quantified to produce 11C-PBR28 distribution volume ratios. These ratios were calculated for the caudate and putamen using the reference Logan plot with the corpus callosum as the reference region. Partial volume effect corrections (Müller-Gartner algorithm) were applied. Differences were sought in Unified Huntington's Disease Rating Scale scores and regional distribution volume ratios between baseline and follow-up and between the two treatment groups (laquinimod versus placebo). No significant change in 11C-PBR28 distribution volume ratios was found post treatment in the caudate and putamen for both those treated with laquinimod (N = 10) and those treated with placebo (N = 5). Over time, the patients treated with laquinimod did not show a significant clinical improvement. Data from the 11C-PBR28 PET-CT study indicate that laquinimod may not have affected regional translocator protein expression and clinical performance over the studied period

Missed Opportunities: Family History and Behavioral Risk Factors in Breast Cancer Risk Assessment Among a Multiethnic Group of Women

BACKGROUND: Clinician’s knowledge of a woman’s cancer family history (CFH) and counseling about health-related behaviors (HRB) is necessary for appropriate breast cancer care. OBJECTIVE: To evaluate whether clinicians solicit CFH and counsel women on HRB; to assess relationship of well visits and patient risk perception or worry with clinician’s behavior. DESIGN: Cross-sectional population-based telephone survey. PARTICIPANTS: Multiethnic sample; 1,700 women from San Francisco Mammography Registry with a screening mammogram in 2001–2002. MEASUREMENTS: Predictors: well visit in prior year, self-perception of 10-year breast cancer risk, worry scale. Outcomes: Patient report of clinician asking about CFH in prior year, or ever counseling about HRB in relation to breast cancer risk. Multivariate models included age, ethnicity, education, language of interview, insurance/mammography facility, well visit, ever having a breast biopsy/follow-up mammography, Gail-Model risk, Jewish heritage, and body mass index. RESULTS: 58% reported clinicians asked about CFH; 33% reported clinicians ever discussed HRB. In multivariate analysis, regardless of actual risk, perceived risk, or level of worry, having had a well visit in prior year was associated with increased odds (OR = 2.3; 95% CI 1.6, 3.3) that a clinician asked about CFH. Regardless of actual risk of breast cancer, a higher level of worry (OR = 1.9; 95% CI 1.4, 2.6) was associated with increased odds that a clinician ever discussed HRB. CONCLUSIONS: Clinicians are missing opportunities to elicit family cancer histories and counsel about health-related behaviors and breast cancer risk. Preventive health visits offer opportunities for clinicians to address family history, risk behaviors, and patients’ worries about breast cancer