Purification and characterization of the carbonic anhydrase enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on enzyme activity

In this study, the carbonic anhydrase (CA) enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney with a specific activity of 603.77 EU/mg and a yield of 35.5% using Sepharose-4B-l-tyrosine- sulphanilamide affinity column chromatography. For determining the enzyme purity and subunit molecular mass, sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The molecular mass of subunit was found approximately 29.71 kDa. The optimum temperature, activation energy (E_a), activation enthalpy (ΔH) and Q₁₀ values were obtained from Arrhenius plot. K_m and V_max values for p-nitrophenyl acetate of the purified enzyme were calculated from Lineweaver-Burk graphs. In addition, the inhibitory effects of different heavy metal ions (Fe²⁺, Pb²⁺, Co²⁺, Ag⁺ and Cu²⁺) on Black Sea trout kidney tissue CA enzyme activities were investigated by using esterase method under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC₅₀) were obtained. Finally K_i values and inhibition types were calculated from Lineweaver-Burk graphs.     

Development of an IgG-Fc fusion COVID-19 subunit vaccine,AKS-452

AKS-452 is a biologically-engineered vaccine comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain antigen (Ag) and human IgG1 Fc (SP/RBD-Fc) in clinical development for the induction and augmentation of neutralizing IgG titers against SARS-CoV-2 viral infection to address the COVID-19 pandemic. The Fc moiety is designed to enhance immunogenicity by increasing uptake via Fc-receptors (FcγR) on Ag-presenting cells (APCs) and prolonging exposure due to neonatal Fc receptor (FcRn) recycling. AKS-452 induced approximately 20-fold greater neutralizing IgG titers in mice relative to those induced by SP/RBD without the Fc moiety and induced comparable long-term neutralizing titers with a single dose vs. two doses. To further enhance immunogenicity, AKS-452 was evaluated in formulations containing a panel of adjuvants in which the water-in-oil adjuvant, Montanide™ ISA 720, enhanced neutralizing IgG titers by approximately 7-fold after one and two doses in mice, including the neutralization of live SARS-CoV-2 virus infection of VERO-E6 cells. Furthermore, ISA 720-adjuvanted AKS-452 was immunogenic in rabbits and non-human primates (NHPs) and protected from infection and clinical symptoms with live SARS-CoV-2 virus in NHPs (USA-WA1/2020 viral strain) and the K18 human ACE2-trangenic (K18-huACE2-Tg) mouse (South African B.1.351 viral variant). These preclinical studies support the initiation of Phase I clinical studies with adjuvanted AKS-452 with the expectation that this room-temperature stable, Fc-fusion subunit vaccine can be rapidly and inexpensively manufactured to provide billions of doses per year especially in regions where the cold-chain is difficult to maintain.     

脑膜癌病的临床与病理特点

目的探讨脑膜癌病的临床与病理特点。方法回顾性分析142例脑膜癌病患者的临床及脑脊液(CSF)细胞学资料和免疫细胞化学标记结果。结果本组患者临床特点:头痛123例(86.62%),头晕19例(13.38%),发热26例(18.31%),视物模糊10例(7.04%)。CSF检查及免疫标记:全部患者CSF中均发现癌细胞;癌细胞上皮膜抗原(EMA)( ),细胞角蛋白(CK)( ),CD14(-),胶质纤维酸性蛋白(GFAP)(-)。其中99例(69.72%)CK低分子( )、高分子(-),诊断为转移性腺癌;33例(23.24%)CK高分子( )、CK低分子(-),诊断为转移性鳞癌;10例(7.04%)CK高分子( )、CK低分子( ),考虑为腺鳞癌。105例找到原发灶,其中67例(47.18%)确诊为肺癌;23例(16.20%)在消化道内找到原发灶;9例(6.34%)为乳腺癌;子宫内膜癌、卵巢癌、肝癌各1例。另有37例(26.06%)未找到原发灶。诊断明确的患者中,15例失访,另90例中有82例(91.11%)于确诊后3～14个月死亡。未找到原发灶的患者中,有6例失访,另31例中有28例(90.32%)在确诊后2～13个月内死亡。结论脑膜癌病患者以头痛、头晕为主要临床表现,并可伴有发热及视物模糊。CSF细胞学检查是诊断脑膜癌病的主要依据,通过免疫细胞化学的方法,可以确定转移癌细胞。脑膜癌病患者预后较差。     

Widespread serum amyloid P immunoreactivity in cortical amyloid deposits and the neurofibrillary pathology of Alzheimer's disease and other degenerative disorders

Amyloid P (AP) component is present in all types of systemic amyloid deposits. Recently, it has been shown to be also present in cerebral amyloid lesions of Alzheimer's disease (AD). In this study, we used immunocytochemical methods to extend these findings at the electron microscope level and characterize the spectrum of AP immunoreactivity in neurofibrillary pathology (NFP) of AD and other neurodegenerative disorders including Down's syndrome (DS), Creutzfeldt-Jakob, Parkinson's, Pick's and diffuse Lewy body diseases and progressive supranuclear palsy. In AD and DS, AP immunoreaction product was evident in all the classical amyloid lesions and NFP in a large sample of all cortical areas examined. The distribution and relative intensity of immunostaining was similar to that of thioflavin S staining in serial sections. In many cases, however, plaques and vessels stained by anti-AP serum were not apparent with thioflavin S. Serial sections immunostained with antiserum to amyloid A, C-reactive protein or to other proteins involved in systemic amyloidoses and the acute phase response showed no evidence of staining in any of the cerebral lesions. Electron microscopy confirmed that AP immunoreactivity was associated with the abnormal filaments characteristic of NFP as well as amyloid fibrils found in plaques and vessels showing congophilic amyloid angiopathy. Plaques of Creutzfeldt-Jakob disease, Pick bodies of Pick's disease, tangles and Lewy bodies in Parkinson's disease and a subpopulation of Lewy bodies in the diffuse Lewy body disease coexistent with AD were also stained. With the exception of vessels in two of the five cases, AP was not detected in age-matched controls. Our observations indicate AP to be a consistent feature of cerebral NFP and amyloid deposits.     

Spindle-cell glioblastoma or gliosarcoma?

'Gliosarcomas' have long been considered to be mixed gliomas and sarcomas. The present study failed to define criteria which clearly delineate 'gliosarcomas' from glioblastoma multiforme and suggests that 'gliosarcomas' should be considered as spindle cell glioblastomas. A total of six cases originally diagnosed as 'gliosarcomas' were compared with four cases of glioblastoma multiforme. No clinical or prognostic features were defined which would clearly separate 'gliosarcomas' from glioblastoma multiforme. Macroscopically, biopsies from 'gliosarcomas' ranged from firm, apparently well-circumscribed tumours to poorly circumscribed lesions with a soft consistency resembling glioblastoma multiforme. Histology revealed a continuous spectrum in which 'gliosarcomas' with large reticulin-rich areas of spindle cells merged with typical glioblastomas containing only small islands of spindle cells and reticulin staining. Immunocytochemistry for glial fibrillary acidic protein (GFAP); S100 protein and alpha-smooth muscle actin (ASMA) showed that the majority of cells in reticulin-poor areas of 'gliosarcoma' and glioblastomas expressed S100 protein and GFAP; many expressed ASMA and some expressed both GFAP and ASMA. Spindle cells in reticulin-rich areas of 'gliosarcomas' and glioblastomas most frequently expressed ASMA but many cells also expressed S100 protein and GFAP; some cells expressed both GFAP and ASMA. The results of this study and a review of the literature suggests that there is a clinical, radiological and pathological continuum with glioblastoma and 'gliosarcoma' at different ends of the spectrum. It is suggested, therefore, that most, if not all, 'gliosarcomas' be redesignated as spindle cell glioblastomas and not be considered as a mixture of glioma and sarcoma.     

Distribution of tyrosine-hydroxylase (TH)-immunoreactive neurons in the diencephalon of the pigeon (Columba livia domestica)

The distribution of tyrosine-hydroxylase (TH)-immunoreactive cell bodies and fibers in the diencephalon has been investigated with immunohistological techniques in the pigeon. The results suggest that TH is present in a number of morphologically distinct neuronal systems. Preoptic and hypothalamic TH neurons were subdivided into a medial periventricular and a lateral group. The medial group starts with a rostral collection of small cells in the preoptic region. A significantly larger collection of TH neurons occupies the paraventricular nucleus (PVN) (stratum cellulare internum) and mainly consists of large multipolar cells. Further caudally, the main concentration of cells is in the hypothalamic posteromedial and the periventricular regions of the tuberoinfundibular (arcuate) nucleus. No TH neuron was found in the ventral and lateral parts of the tuberoinfundibular region, suggesting that the prominent tuberoinfundibular dopaminergic system described in mammals is absent in the pigeon. This further substantiated by the relative scarcity of TH immunoreactive fibers and varicosities in the neurohemal zone of the median eminence (ME). The caudalmost components of the medial group appear to be continuous with the large population of TH neurons distributed in the midline of the mesencephalon. Tyrosine-hydroxylase-immunopositive cells have not been found in the paraventricular organ. The lateral group consists of TH neurons loosely arranged in the lateral hypothalamus, including regions of the supraoptic nucleus and hypothalamic posterolateral nucleus. Tyrosine-hydroxylase containing neurons vary widely in size, shape, and dendritic arborization in each diencephalic region. However, it is possible to distinguish two main cell types. Small bipolar neurons with two simple arborizing dendrites were concentrated in the medial periventricular system. The second type of cell is large, multipolar with four to five branching dendrites. This latter cell type occurs mainly in the lateral system and in the PVN. Major fiber bundles containing TH immunoreactivity were identified in the lateral and periventricular hypothalamus. The paraventricular organ and the organum vasculosum laminae terminalis contained the densest arborization of fibers and varicosities. In the ME, dense innervation was found in the subependymal layer. Dense arborizations of TH positive fibers and varicosities were located in the septal nuclei and the paleostriatum augmentatum.     

Extracellular Enzyme Activities by Basidiobolus and Conidiobolus Isolates on Solid Media./Extrazelluläre Enzymaktivitäten bei Basidiobolus- und Condiobolus-Isólaten auf festen Medien

Summary: Fifty-seven isolates of Basidiobolus from reptiles and amphibians, and 7 other obtained from the American Type Culture Collection (ATCC) in Maryland were studied for their extracellular enzyme activities on solid media. The Conidiobolus isolates studied included 4 recovered from Nigerian soil and additional 4 obtained from the ATCC All these isolates produced active extracellular lipase and protease and failed to exhibit amylase, deoxyribonuclease and ribonuclease activities. The significance of the findings is discussed.
Zusammenfassung: Fünfundsiebzig Basidiobolus-Isolate aus Reptilien und Amphibien und sieben weitere aus der American Type Culture Collection (ATCC) in Maryland wurden auf Aktivtäten extrazellulärer Enzyme auf festen Medien untersucht Die untersuchten Conidiobolus-Isolate schlossen vier aus nigerianischen Böden und vier weitere aus der ATCC ein. Alle Isolate zeigten extrazellulär Lipase- und Proteaseaktivität Amylase-, Desoxyribonuclease- und Ribonucleaseaktivität war jedoch nicht nachzuweisen. Die Bedeutung dieser Befunde wird diskutiert.     

日本血吸虫酚氧化酶的组化定位研究

目的观察酚氧化酶(phenol oxidase,PO)在日本血吸虫成虫的组织学分布.方法酚氧化酶组化方法:将42 d虫龄的日本血吸虫活成虫置含25 mmol/L邻苯二酚的磷酸盐缓冲液(pH 6.8)中,37℃孵育30 min后,转移虫体至载玻片上,吸去虫体表面液体,将其中一部分虫体置Olym-pus显微镜下观察酚氧化酶在虫体的组织学分布.荧光组织化学方法:经上述过程处理后,在另一部分虫体上滴加2滴含0.05%戊巴比妥钠的PBS溶液,然后置Leica荧光显微镜下观察酚氧化酶在虫体的组织学分布.结果酶组织化学方法显示,日本血吸虫酚氧化酶仅分布于雌虫卵黄腺及子宫内虫卵卵壳表面,呈现棕褐色显色反应;雌虫卵巢和雄虫均未发现酚氧化酶活性.然而,荧光组织化学方法显示,酚氧化酶除主要分布于雌虫卵黄腺及子宫内虫卵卵壳表面,呈现强荧光外,还少量分布于雄虫体壁表层,呈现弱荧光反应.结论不仅日本血吸虫雌虫含有酚氧化酶活性,而且雄虫也含酚氧化酶活性,只不过其含量少、活性低.荧光组织化学方法能更灵敏地显示日本血吸虫酚氧化酶活性,更适用于日本血吸虫酚氧化酶的组织学定位.