Introduction of marine-derived Streptomyces sp. UTMC 1334 as a source of pyrrole derivatives with anti-acetylcholinesterase activity.

AIM: Alzheimer's disease (AD) is the most common cause of dementia. The acetylcholinesterase (AChE) inhibitors are the most viable therapeutic target for its symptomatic treatment. The present study was aimed at exploring anti-AChE metabolite producing marine Actinobacteria. METHODS AND RESULTS: Of 220 isolates, 34 Actinobacteria extracts were tested for the presence of AChE inhibitors. The obtained results showed that bacterial strain UTMC 1334, inhibited AChE activity in a dose-dependent manner (IC50  = 0·36 ± 0·019 mg ml-1 ). Based on anti-oxidant and cytotoxicity studies, the most potent extract was able to scavenge DPPH radicals with an IC50 value of 45·67 µg ml-1 with the least cytotoxicity. The GC-MS analysis demonstrated that the mentioned activities could be related to pyrrole-derived compounds as were found the predominant constituents in the extract. The most active extract belonged to the strain that shows 99·41% similarity with Streptomyces lateritius based on 16S rRNA gene sequencing. CONCLUSIONS: Our results show anti-AChE activity is prevalent in marine Actinobacteria, and even in rather comparable prevalence with the antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights that exploring new lead anti-AChE compounds may result in discovering novel adjuvant candidates with potency in the treatment of cognitive diseases such as AD.

Catechol alkenyls from Semecarpus anacardium: acetylcholinesterase inhibition and binding mode predictions.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Semecarpus anacardium L. f. (Anacardiaceae) are used in Ayurvedic medicine and also in Iranian Traditional Medicine for various indications, among those for retarding and treatment of dementia. AIM OF THE STUDY: The severity of Alzheimer's disease obviously correlates with a cholinergic deficit. In a screening for acetylcholinesterase (AChE) inhibitory activity, an extract from the fruit resin of Semecarpus anacardium was among the most active ones. Thus, the aim of this study was to isolate the active compounds and to investigate them in detail. Their binding mode to the active site of AChE was investigated by in silico docking experiments. MATERIALS AND METHODS: From a dichloromethane extract in an activity-guided fractionation the active compounds were isolated under use of different chromatographic techniques. Their structures were unambiguously identified by one and two-dimensional (1)H and (13)C NMR spectroscopy and mass spectrometry and their cholinesterase inhibitory activities were determined by a microplate assay. In order to compare the 3D active sites of AChE from Torpedo californica (TcAChE) and from Electrophorus electricus (EeAChE), three files from the Protein Data Bank (PDB) were used and for docking experiments, GOLD 3.1 software was employed. The concentrations of active compounds in the extract and the fruits were determined by HPLC analysis. RESULTS: The active compounds were determined as 1',2'-dihydroxy-3'-pentadec-8-enylbenzene (A) and 1',2'-dihydroxy-3'-pentadeca-8,11-dienylbenzene (B). Their IC(50) values in an in vitro assay on AChE inhibition were determined as 12 and 34 µg/mL, respectively, while they were not active in the inhibition of butyrylcholinesterase (BChE). In silico docking experiments showed a similar bioactivity for compounds A and B. The concentration of compounds A and B in the fruits was 1.85% and 1.88%, respectively. CONCLUSION: In the search for the active principle of the fruit resin of Semecarpus anacardium, compounds A and B were identified as two selective inhibitors for AChE versus BChE.

Influence of 6-mercaptopurine on the prenatal development of the rat cortex.

The prenatal cortex development after the application of 6-Mercaptopurine (6-MP) was studied in the rat embryo. 24 h after application of a single dose of 6-MP on day 12, 13, 14 or 15 of gestation first cell lesions occured in the form of necroses. 48 h after injection of the drug these necroses were most pronounced. Their extent and distribution pattern was, however, dependent on the developmental stage. In the early developmental phase, on days 12 and 13, massive necroses could be observed in the intermediate zone. At later stages of development (drug application on day 14 or 15) damaged cells could occasionally also be found scattered over the whole ventricular wall. The late toxic response to 6-MP treatment suggests that, after incorporation of metabolites of the agent into DNA during the S-phase, at least one additional S-phase is completed before the disturbance created in a cell is sufficient to cause necrosis. The necrosis may be a result of an action of 6-MP directly on DNA or via an affect on transcription. The different extent of the lesion during the various developmental phases suggests different growth dynamics of the cortex.

[Linear- and Areal-analytical results of the karyometric studies on monolayer cultures and the influence of actinomycin (author's transl)]. / Linear- und flächenanalytische Ergebnisse karyometrischer Untersuchungen an Monolayerkulturen unter dem Einfluss von Actinomycin D

Actinomycin D (AMD) inhibits the RNA-synthesis by intercalating with the DNA-helix. In monolayer culture of embryonic mouse brain cells in the 8th passage the cell nuclei were demonstrated with the Feulgen-reaction and quantitatively-morphometrically evaluated following a 48 h intoxication with AMD (0.001; 0.01; 0.1 mug/ml). The mean value of nuclear area calculated from single measurements with the Quantimet 720 is significantly larger when compared to the controls at a dose of 0.01 mug AMD/ml. At 0.1 mug AMD/ml ca. 30% of the cell nuclei are pyknotic; the non-pyknotic nuclei are enlarged significantly by appr. 20%. The linear-analytic results using a Digiscan show no enlargement of tendon length, on the contrary, at 0.1 mug AMD/ml a significant diminution versus the controls can be seen. This resutls from the pyknotic cell nuclei which are also measured in this case no single measurements were carried out. The determination of cell density using a Digiscan shows a diminution of cell number by 19, 31 or 68% dependent upon AMD-concentration.

Influence of cell density of nuclear size in monolayer cells from embryonic mouse brains.

Studies were carried out on the nuclear size of embryonic mouse brain monolayer cells at various cell densities. The variation in cell density resulted from different initial cell numbers. The nuclear area was measured area-analytically with the Quantimet, the determination of cell population was measured linear-analytically using the Digiscan. Nuclear size remains constant in a cell culture of 10(4) up to 8.3 X 10(4) cells ml, which corresponds to a cell density of about 25-160 cells/unit. A significant diminution in mean nuclear size of the cell nucleus occurs in the case of increased cell densities of 215 and 277 cells/unit, corresponding to a cell culture of 2.5 X 10(5) AND 7.3 X 10(5) cells ml. This diminution may be connected with the accumulation of G1 cell nuclei in cultures with contact-dependent growth inhibition.

Morphometric studies on the relationship between myelin sheath and axon area in the sciatic nerve of adult rats.

The myelin sheath and axon areas in the sciatic nerve of 20 adult rats were studied using the video-point-counting method (Quantimet). This automatic measurement procedure allows an exact and rapid determination of the above mentioned areas as a result of the differing intensities of the structures. The electron-microscopic pictures are projected at a magnification of 3500 onto the monitor through a lens system from a Vidikon-TV-tube, objective 32 mm. The areas of single nerve cross-sections, myelin sheath areas and total area are then measured. These area values are stated in picture points which can be calculated to absolute values. The statistical evaluation of both parameters shows a linear regression with a correlation coefficient of 0.94. These values are compared with the results obtained using the conventional manual methods of other authors, e.g. determination of lamellae number or myelin sheath thickness as well as axon circumference or axon diameter. The special advantages of this procedure are thereby pointed out.

Electronmicroscopic study of the effect of 6-an on the sciatic nerve in newborn rats.

12 hrs, 1, 2, and 4 days after i.p. administration of 2, 3 or 5 mg/kg 6-AN (6-Aminonicotinamide) on postnatal day 5, the following findings were made in the sciatic nerve of the newborn rat. In the myelin-forming fibres a swelling between the axolemm and the first inner intraperiod-line of the Schwann cell develops with displacement of the otherwise intact axon. The myelin formation is not impeded. During the 4 days of control after administration of 6-AN there is continual growth of the myelin sheath with an orderly lamellar structure. Using any dose there is no demonstrable alteration in the Schwann cells--with the exception of the inner cytoplasmic tongue--either in the foetal promyelin and mature nonmyelinated fibres. These findings are discussed and compared with the known glia alterations of the spinal ganglia and dorsal radix after the application of 6-AN.

Histological effects of 6-mercaptopurine on the fetal rat central nervous system: a light-microscopic study.

Wistar rats were administered single doses of 16 or 50 mg/kg 6-mercaptopurine (6-MP) on day 12 of pregnancy. Necrosis in the fetal forebrain and spinal cord was studied by light microscope 6, 12, 14, 48, 72, and 81 h and 8 days afterward. The extent of necrosis was dose dependent. The first necroses were seen after 24 h, regardless of location (brain, spinal cord) or dose; but the extent was greatest after 48 h. All necrotic cells had a typical appearance; they were ballooned and often fragmented, their nuclei were darkly colored and frequently pyknotic, and they were often karyorhexic. Necroses appeared almost exclusively at sites of beginning cellular differentiation, i.e., in the intermediate zone. In the spinal cord the ventricular zone was also necrotic and the alar plate (dorsal horn) always affected. Phagocytizing cells (macrophages) appeared in the spinal cord after 48 h and in the brain after 72 h. After 81 h all the necrotic material had been phagocytized, at which time there was a massive congestion of the extra- and intracerebral vessels. Hemorrhages appeared in defined localizations. Eight days after exposure to 16 mg/kg 6-MP fetuses no longer showed any visible deviations. Fetuses exposed to 50 mg/kg showed deviations in the cytoarchitecture of the neopallium: an extremely broadened ventricular zone, few cells in the intermediate zone, and extensive rarefaction cells in the cortical plate with no clear layer structure. In the spinal cord, cleft formations were especially noticeable in the dorsal-horn region. All fetuses showed a hydrocephalus externus after 50 mg/kg. The mechanism leading to necrosis is discussed.

Change of nuclear size in monolayer cells from embryonic mouse brain under the influence of cytosine-arabinoside.

The influence of cytosine-arabinoside (ara-C) on the nuclear size of monolayer cells from mouse brains were studied by quantitative analysis with the Quantimet (area measurement) and linear analysis with the Digiscan. Ara C (0,1; 1,0; 10,0 mug/ml) causes a significant nuclear enlargement, depending on the dose of 16%, 30% and 35% respectively and at the same time a decrease in cell density and an inhibition of mitotic activity. The size-distribution graph is altered by ara-C: the graph flattens and widens at the base. Moreover, in the case of 10,0 mug ara-C/ml a pronounced graph shoulder is visible in the small nuclear size region which can be interpreted as a slight accumulation of small nuclei. The nuclear enlargement is possibly due to an accumulation of shortchain polynucleotides.

[Measurement of nuclear size in cells from embryonic mouse brain in monolayer culture using automatic image analysers]. / Messung von Kerngrössen an Zellen von embryonalen Mäusegehirnen in Monolayer-Kultur mit automatischen Bildanalysegeräten

Size analyses and size classifications were made of the nuclei of embryonal mouse brain cells in monolayer culture. Measurements were made by surface analysis, using the Quantimet (1,299 nuclei), and linear analysis, using the Digiscan (1,887 nuclei). Despite the different methods of measurement and different logarithmic classification, similar distribution curves were obtained, approaching the Gaussian normal distribution. The methodical problems of karyometry as well as the problems of statistical data processing are discussed.

[The use of stereologic image analysis in medicine (peripheral nerves)]. / Uber die Anwendung der stereologischen Bildanalyse in der Medizin (Periphere Nerven)

The use of image plane scanning for the purpose of quantification of data is becoming increasingly important in medicine and biology. Such equipment allows complex geometric-statistical relationships to be investigated by reduction to a counting process in which picture elements are counted (using a photocell), stored, and evaluated. Various absolute values of interest can then be computed using certain mathematical relationships. These methods, using automatically controlled sampling, are fast, and, since they are able to detect small variations in light intensity, very accurate. Furthermore, even in the presence of highly complex patterns, an accurate surface area measurement can be made. Peripheral nerve cells, for example, lend themselves well to such methods, since the strong contrast between individual structural elements provides excellent conditions for automatic measurement.