Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis

Recent experimental evidence indicates that non-neuronal acetylcholine is involved in the regulation of basic cell functions. Here we investigated the cholinergic system in the skin of healthy volunteers and patients with atopic dermatitis (AD). The synthesizing enzyme, choline-acetyltransferase (ChAT), was studied by anti-ChAT immunohistochemistry and enzyme assay. Skin biopsies taken from healthy volunteers and from AD patients were separated into the 2 mm superfical (epidermis and upper dermis) and 3 mm underlying portion (deeper dermis and subcutis). ChAT enzyme activity was detected in homogenized skin and subcutaneous fat (about 13 nmol/mg protein/h). ChAT immunoreactivity was expressed in keratinocytes, hair papilla, sebaceous and eccrine sweat glands, endothelial cells and mast cells. In healthy volunteers the superficial and underlying portion of skin biopsies contained 130 +/- 30 and 550 +/- 170 pmol/g acetylcholine (n = 12), respectively. In AD patients (n = 7) acetylcholine was increased 14-fold in the superficial and 3-fold in the underlying biopsy portion. The present study demonstrates the widespread expression of ChAT protein in the vast majority of human skin cells. Tissue levels of acetylcholine are greatly (14-fold) enhanced in the superficial 2 mm skin of AD patients.     

Significance of histiocytes on otherwise-normal cervical smears from postmenopausal women. A retrospective study of 108 cases

OBJECTIVE: To evaluate the significance of histiocytes on normal cervical smears from postmenopausal women and correlate them with endometrial pathology. STUDY DESIGN: Histiocytes were classified into three types. The clinical history was obtained from cytologic and surgical reports. RESULTS: Among 108 cervical smears, 13 had large, foamy histiocytes (type A), 88 had histiocytes resembling superficial endometrial stromal cells (type B), and 7 had variably sized histiocytes alone or in association with inflammatory or multinucleated cells (type C). Endometrial pathology was identified in 13 patients (12.0%): 4/13 with type A histiocytes (2 endometrial adenocarcinomas, 2 endometrial polyps), 8/88 with type B histiocytes (8 endometrial polyps) and 1/7 with type C histiocytes (endometrial polyp). Among 70 patients with no clinical indications for endometrial sampling except for the presence of histiocytes, 4 demonstrated endometrial pathology (all endometrial polyps). In contrast, endometrial pathology was identified in 9/38 with clinical indications for endometrial sampling. Among the 13 patients with endometrial pathology, 9 had a significant clinical history (sensitivity of 69.2%), and 4 had histiocytes as the only indication for endometrial biopsy (sensitivity of 30.8%). CONCLUSION: A significant clinical history is more predictive of endometrial pathology and outweighs the significance of histiocytes as an indication for endometrial biopsy.     

Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions

The budding yeast spindle aligns along the mother- bud axis through interactions between cytoplasmic microtubules (CMs) and the cell cortex. Kar9, in complex with the EB1-related protein Bim1, mediates contacts of CMs with the cortex of the daughter cell, the bud. Here we established a novel series of events that target Kar9 to the bud cortex. First, Kar9 binds to spindle pole bodies (SPBs) in G(1) of the cell cycle. Secondly, in G(1)/S the yeast Cdk1, Cdc28, associates with SPBs and phosphorylates Kar9. Thirdly, Kar9 and Cdc28 then move from the SPB to the plus end of CMs directed towards the bud. This movement is dependent upon the microtubule motor protein Kip2. Cdc28 activity is required to concentrate Kar9 at the plus end of CMs and hence to establish contacts with the bud cortex. The Cdc28-regulated localization of Kar9 is therefore an integral part of the program that aligns spindles.     

Role of the spindle pole body of yeast in mediating assembly of the prospore membrane during meiosis

Spindle pole bodies (SPBs) are the centrosome equivalents in yeast, required for microtubule organization. In yeast, the SPB further serves as the attachment sites of the prospore membrane during meiosis. Here we report the identification of two new meiosis-specific components of the SPB, Mpc54p and Mpc70p, and the first protein specific for the prospore membrane, Don1p. Mpc54p and Mpc70p are not present in mitotic SPBs, and during meiosis II they are components of a meiosis-specific structural alteration of the outer plaque of the SPB. Both proteins are dispensable for the meiotic divisions but are essentially required for the formation of the prospore membrane. In the mpc54 and mpc70 mutants, the Don1p-containing precursors of the prospore membrane can still be found in the cytoplasm and associated with the SPB. Unexpectedly, however, the assembly of the precursors to a continuous membrane system is affected. Thus, the meiotic SPB is directly involved in the formation of a specialized membrane system, the membrane of the prospore.     

The largest moss carpet transplant in Antarctica and its bryosphere cryptic biodiversity

Extremophiles - As part of the reconstruction of the Brazilian Antarctic Station on King George Island, three areas of moss carpet were transplanted to minimize the impact of the new facilities on...     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.     

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals¹. Though being a crucial part of the visual processing by shaping the retinal output², most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue^3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage⁵. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments⁶.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.