Endopeptidase activities of selected Porphyromonas spp., Prevotella spp. and Fusobacterium spp. of oral and non-oral origin

The ability of three Porphyromonas spp., seven Prevotella spp., seven Fusobacterium spp. and two related Bacteroides spp. (B. levii and B. macacae) to degrade an extensive range of synthetic endo-, amino- and diamino peptidase substrates linked to the fluorescent leaving group 7-amido-4-methylcoumarin (NHMec) was investigated. Many more species than was previously recognized exhibited peptidase activities, albeit at lower levels than those already described for Porphyromonas gingivalis. Detection of chymotrypsin-like activity was dependent on which of three NHMec-linked substrates was used, but all species exhibited degradative activity with at least one of these substrates. Elastase-like activity was detected in all species though not all species reacted with each of the elastase substrates. Glycylprolyl peptidase activity was detected in all of the species tested with the exception of F. mortiferum, F. gonidiaformans, F. naviforme and F. necrophorum. While the detection of peptidase activities does not appear to be useful for the differentiation of species within the genera Bacteroides and Prevotella, its ability to differentiate species of the genus Porphyromonas or Fosobacterium further investigation.     

Acetylcholine Synthesis in a Primary Culture of Porcine Intermediate Lobe Cells

Previous pharmacological studies with the pituitary gland have suggested that acetylcholine (ACh) might be involved in the regulation of intermediate lobe (IL) function. Whether ACh is endogenous to the IL cells or provided from an extrinsic source is unclear. The present experiments tested the possibility that the endocrine cells of the IL may be a source of ACh by measuring certain cholinergic markers in a primary culture of dissociated porcine cells. The endogenous ACh content was readily measurable in both the freshly dissociated IL cells and in 2- or 4-day primary cultures. Choline acetyltransferase activity was also measurable in the freshly dissociated and cultured IL cells and was reduced by 53% in the presence of a specific inhibitor, napthylvinylpyridine (50 μM). IL cells incubated in the presence of [¹⁴C]choline (1 μM) were able to synthesize [¹⁴C]ACh and the accumulation of the new ACh was inhibited by hemicholinium-3 (30 μM), a competitive inhibitor of high affinity choline uptake at cholinergic nerve terminals. In conclusion, these results demonstrate that the endocrine cells of the IL are capable of synthesizing and storing ACh.     

Polysialylated Neural Cell Adhesion is Involved in Target-induced Morphological Differentiation of Arcuate Dopaminergic Neurons

We have previously shown that the morphological and biochemical maturation of developing rat hypothalamic dopaminergic neurons is accelerated when they are cocultivated with pituitary intermediate lobe cells, one of their targets. Only two subsets of hypothalamic dopaminergic neurons (arcuate, A12, and periventricular, A14, nuclei) may project to the pars intermedia. In order to determine whether the two populations are equally responsive to coculture conditions, we microdissected the hypothalamus of 17-day-old rat fetuses in two fragments containing cell bodies from the A12 and from the A14 regions, prepared neuronal cultures from both portions and incubated them separately with intermediate lobe cells. The presence of intermediate lobe cells increased tyrosine hydroxylase levels in both dopaminergic neuron subsets, but morphological differentiation was accelerated in dopaminergic neurons originating in the arcuate nucleus only. We then investigated whether physical contact between developing arcuate neurons and their target cells was a prerequisite of the morphological effect by interposing a semipermeable membrane between cultivated neurons and intermediate lobe cells in transwell culture dishes. The morphological effect was no longer observed under transwell coculture conditions, pointing to the involvement of membrane-bound molecules. Accordingly, the stimulating effect of coculture on arcuate dopaminergic neurons was completely abolished by the removal of polysialic acid on neural cell adhesion molecules by endoneuraminidase N treatment. Thus, maturation of A12 and A14 dopaminergic neurons exhibits differential susceptibility to intermediate lobe target cells, and polysialylated-NCAM is required for the contact-dependent effect.     

Characterisation of black-pigmented anaerobes isolated from diseased and healthy periodontal sites

Prevotella intermedia has recently been re-defined and a new species, Prevotella nigrescens has been proposed. However, there is little data available on the incidence of these new species in periodontal health or disease. Black-pigmented anaerobes isolated from diseased and healthy subgingival sites were identified by serotyping, SDS-PAGE and physiological tests. In adult periodontitis subjects, 64% of active sites, 35.7% of inactive sites and 38.5% of healthy sites yielded black-pigmented anaerobes. Of these, Porphyromonas gingivalis was found in 11% of active and 5% of healthy sites in diseased patients, Prevotella intermedia in 15.5% of active and 20.5% of healthy sites, Prevotella nigrescens in 37.7% of active and 11.5% of healthy sites and Prevotella denticola in 3% of active and 1% of healthy sites. In healthy subjects, 50% of sites yielded black-pigmented anaerobes. P. gingivalis was not found in healthy subjects but P. intermedia was found in 18% and P. nigrescens in 31% of sites. SDS-PAGE proved to be a useful method for routinely differentiating P. intermedia and P. nigrescens and two sub-types of the latter species were detected on the basis of band pattern. Only one P. nigrescens sub-type was found in any given individual and one type, typified by ATCC 25261, was more commonly found in deep pockets. However, overall both P. nigrescens and P. intermedia as species were just as frequently found at healthy sites as diseased sites. Thus, these species, in contrast to P. gingivalis , appear to be common commensals but they may act as opportunistic pathogens.     

Nitric oxide synthase and background adaptation in Xenopus laevis

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by α-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). α-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain.In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background.Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3′,5′ guanosine monophosphate (cGMP)-accumulating fibers in the PI.The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.     

新疆中麻黄DNA片段的克隆与序列分析

对新疆3种中药麻黄(Ephedra)进行了RAPD分析，回收随机引物s13的PCR产物(750bp左右)，克隆到pMD18-T载体中，经电泳鉴定后进行序列分析。结果表明，已成功克隆并获得了新疆中麻黄771bp的DNA片段，对中药材的鉴定和标准化提供依据。     

Identification of the Asymmetric Hybrid of Human Oxy Hemoglobins A and S (α2βAβS) at 4° C by Cellulose Acetate Electrophoresis

Millions of people are affected by hereditary hemochromatosis (HH) and thalassemia intermedia (TI), the iron overloading disorders caused by chronic increases in iron absorption. Genetic factors, regulatory pathways involving proteins of iron metabolism, non regulatory molecules, dietary constituents and iron binding drugs could affect iron absorption and could lead to iron overload or iron deficiency. Chelators and chelating drugs can affect both iron absorption and excretion. Deferoxamine (DFO), deferiprone (L1) and the DFO/L1 combination therapies have been used effectively for reversing the toxic side effects of iron overload including cardiac and liver damage in TI and HH patients where venesection is contraindicated. Selected protocols using DFO, L1 and their combination could be designed for optimizing chelation therapy in TI and HH. The use of deferasirox (DFRA) in HH and TI could cause an increase in iron and other toxic metal absorption. Future treatments of HH and TI could involve the use of iron chelating and other drugs not only for increasing iron excretion but also for preventing iron absorption.     

A Clinical Update of the Hb Siirt [β27(B9)Ala→Gly; HBB: c.83C>G] Hemoglobin Variant

We report a clinical update of the hemoglobin (Hb) variant [β27(B9)Ala→Gly; HBB: c.83C>G], named Hb Siirt, that was previously described as a silent variant in a 23-year-old Kurdish female. The patient was also a carrier of the codon 5 (–CT) (HBB: c.17_18delCT) frameshift mutation and of the ααα ^{anti 3.7} triplication. Her initial moderate β-thalassemia intermedia (β-TI) phenotype worsened with time, causing the patient to become a transfusion-dependent subject at the age of ～40 years. Subsequent molecular characterization of both parents revealed that the Hb Siirt variant was inherited by the mother, while the other two globin alterations (HBB: c.17_18delCT and ααα^{anti 3.7} triplication) were genetically transmitted by the father. The latter remained a carrier of a mild β-TI phenotype throughout his life, at least until the age of 65 years. We hypothesize that the worsened clinical conditions in the daughter were due to the additional, maternally inherited Hb Siirt variant. However, protein 3D conformational analysis did not seem to reveal substantial overall structural changes. Among the other three described variants [Hb Volga (HBB: c.83C>A), Hb Knossos (HBB: c.82?G>T), Hb Grange-Blanche (HBB: c.83C>T] that are due to nucleotide substitutions at codon 27 of the β-globin gene; only Hb Knossos causes a β⁺-thalassemia (β⁺-thal) phenotype.     

Erythrocytic phosphatidylserine exposure and hemostatic alterations in β-thalassemia intermediate patients

Introduction

Hypercoagulable state is one of the common findings in beta-thalassemia intermedia (β-TI), particularly in splenectomized patients, with infrequent blood transfusion. Abnormality of the red blood cells (RBC) membrane due to oxidative damage is suggestive of possible etiologies. Membrane lipid peroxidation increases the exposure of phosphatidylserine (PS) that plays a role in the activation of coagulation factors V and X, subsequently initiating thrombosis. Our aim of this study was to find the probable correlation of the alteration of the PS on the RBC outer membrane with the hypercoagulable state in the β-TI patients.

Materials and methods

Our cross-sectional study was conducted on 39 splenectomized β-TI patients and 38 age-matched healthy controls. The mean age was 37 years. Analysis of the PS exposure on the RBCs was performed by fluorescein isothiocyanate (FITC) conjugated AV protein .Measurement of the coagulation factors X, V and antithrombin III (AT-III) was performed. We also checked the D-dimer levels .Analysis was performed by SPSS16.

Results

Fluorescence of FITC-Annexin V labeling on patients RBCs were higher than healthy controls; (2.8 ± 2.2%) of the patients versus (0.4 ± 0.18%) in the control group and was statistically significant (P < 0.05). Mean levels of factor X and AT-III of the patients as compared with the control group decreased and showed significant difference (P < 0.05).

Conclusions

Circulation of thalassemic RBCs, which abnormally possess PS on RBC membrane outer surface, suggests the possibility of the gradual consumption of the coagulation factors in the presence of a chronic coagulability state.