The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5(H) and D6(H) are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system

Resolution of Ultramicroscopy and Field of View Analysis

In a recent publication we described a microscopical technique called Ultramicroscopy, combined with a histological procedure that makes biological samples transparent. With this combination we can gather three-dimensional image data of large biological samples. Here we present the theoretical analysis of the z-resolution. By analyzing the cross-section of the illuminating sheet of light we derive resolution values according to the Rayleigh-criterion. Next we investigate the resolution adjacent to the focal point of the illumination beam, analyze throughout what extend the illumination beam is of acceptable sharpness and investigate the resolution improvements caused by the objective lens. Finally we conclude with a useful rule for the sampling rates. These findings are of practical importance for researchers working with Ultramicroscopy to decide on adequate sampling rates. They are also necessary to modify deconvolution techniques to gain further image improvements

Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

Semi-Automated Reconstruction of Neural Processes from Large Numbers of Fluorescence Images

We introduce a method for large scale reconstruction of complex bundles of neural processes from fluorescent image stacks. We imaged yellow fluorescent protein labeled axons that innervated a whole muscle, as well as dendrites in cerebral cortex, in transgenic mice, at the diffraction limit with a confocal microscope. Each image stack was digitally re-sampled along an orientation such that the majority of axons appeared in cross-section. A region growing algorithm was implemented in the open-source Reconstruct software and applied to the semi-automatic tracing of individual axons in three dimensions. The progression of region growing is constrained by user-specified criteria based on pixel values and object sizes, and the user has full control over the segmentation process. A full montage of reconstructed axons was assembled from the ∼200 individually reconstructed stacks. Average reconstruction speed is ∼0.5 mm per hour. We found an error rate in the automatic tracing mode of ∼1 error per 250 um of axonal length. We demonstrated the capacity of the program by reconstructing the connectome of motor axons in a small mouse muscle

Expression of UV-Sensitive Parapinopsin in the Iguana Parietal Eyes and Its Implication in UV-Sensitivity in Vertebrate Pineal-Related Organs

The pineal-related organs of lower vertebrates have the ability to discriminate different wavelengths of light. This wavelength discrimination is achieved through antagonistic light responses to UV or blue and visible light. Previously, we demonstrated that parapinopsin underlies the UV reception in the lamprey pineal organ and identified parapinopsin genes in teleosts and frogs of which the pineal-related organs were reported to discriminate light. In this study, we report the first identification of parapinopsin in the reptile lineage and show its expression in the parietal eye of the green iguana. Spectroscopic analysis revealed that iguana parapinopsin is a UV-sensitive pigment, similar to lamprey parapinopsin. Interestingly, immunohistochemical analyses using antibodies specific to parapinopsin and parietopsin, a parietal eye green-sensitive pigment, revealed that parapinopsin and parietopsin are colocalized in the outer segments of the parietal eye photoreceptor cells in iguanas. These results strongly suggest that parapinopsin underlies the wavelength discrimination involving UV reception in the iguana parietal eye. The current findings support the idea that parapinopsin is a common photopigment underlying the UV-sensitivity in wavelength discrimination of the pineal-related organs found from lampreys to reptiles

Tissue Clearing and Deep Imaging of the Kidney Using Confocal and Two-Photon Microscopy

Microscopic and macroscopic evaluation of biological tissues in three dimensions is becoming increasingly popular. This trend is coincident with the emergence of numerous tissue clearing strategies, and advancements in confocal and two-photon microscopy, enabling the study of intact organs and systems down to cellular and sub-cellular resolution. In this chapter, we describe a wholemount immunofluorescence technique for labeling structures in renal tissue. This technique combined with solvent-based tissue clearing and confocal imaging, with or without two-photon excitation, provides greater structural information than traditional sectioning and staining alone. Given the addition of paraffin embedding to our method, this hybrid protocol offers a powerful approach to combine confocal or two-photon findings with histological and further immunofluorescent analysis within the same tissue

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.United States. National Institutes of Health (1-U01-NS090473-01