bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Structure-Function Relationships of the Unique Proteinase Inhibitor Human α2 Macroglobulin
Authors: Stoops James K , Kolodziej Steven J , Qazi Usman , Nolasco Norman J , Gettins Peter GW , Strickland Dudley K 
Published: July 2020

Abstract
Human α2 macroglobulin (α2M) has structural and functional properties that contribute to its uniqueness as proteinase inhibitor. It is the largest known (Mr=720,000) and the only natural proteinase inhibitor which has a broad range of reactivity and for which the reaction is irreversible. It has a vital role in the clearance of proteinases from the circulation and in regulating their activity in fibrinolysis, coagulation and complement activation.
An α2M molecule can entrap two proteinase molecules such as chymotrypsin and trypsin and can therefore be considered to contain two functional domains. Each subunit in the homotetramer has a bait region with cleavage sites for nearly all known endoproteinases and an internal thiol ester bond. A proteinase cleaves the two bait regions within both functional units leading to an activation and cleavage of the thiol ester bonds. Consequently, α2M undergoes a major structural change resulting in the irreversible entrapment of the proteinase.
Fluoronanogold: An Efficient Labeling Reagent for Immunocytochemistry
Authors: Robinson JM 
Published: July 2020

Abstract
The field of immunocytochemistry comprises a large body of methodologies whose purpose is to obtain spatial and temporal information about biological samples with a high degree of chemical specificity. The major differences in immunocytochemical methods are: (1) type of sample preparation (i.e., pre-embedding or post-embedding) and (2) type of detection system (i.e., chromogenic, fluorochromes, or particulate). The history of advances in immunocytochemistry can be understood, to a large extent, through the development of the various detection systems. All of these labeling systems have their appropriate applications and are in use today.
The introduction of colloidal gold as a particulate immunoprobe revolutionized immunocytochemistry, particularly at the ultrastructural level. The fact that these probes are discrete particles that are electron dense contributes to their wide acceptance. Due to the particulate nature of these probes, labeling density over a given structure or region of a cell can be determined by simple counting procedures.
Fluorescent Antenna Proteins from the Bioluminescent Bacteria
Authors: Lee John 
Published: July 2020

Abstract
The emission spectrum from bioluminescent bacteria has been observed to depend on the type of bacteria. Photobacterium phosphoreum species usually show bioluminescence maxima around 472 nm and Photobacterium leiognathi species to slightly longer wavelength. A certain strain (Yl) of Vibrio fischeri, has a yellow bioluminescence with maximum at 542 nm. These differences have been explained as due to the bioluminescence originating from the fluorescence transition of an “antenna” protein, participating in the bioluminescence reaction along with the enzyme bacterial luciferase. The bioluminescence from a number of coelenterates involves a similar participation of an antenna protein, the famous “Green-Fluorescent Protein” being the origin of the bioluminescence emission from these organisms.
Quantitative Imaging Of the Green Fluorescent Proteins
Authors: Piston David W , Patterson George H , Knobel Susan M 
Published: July 2020

Abstract
The cloning and expression of GFP in heterologous systems introduced a fantastic tool for studying specific gene expression and protein localization inside living cells. However, one aspect of GFP that has not been exploited to its full potential is its use as a quantitative imaging tool. To determine its quantitative usefulness, we have addressed five points that are important in GFP imaging: detectable signal over background, photostability, pH stability of the molecule, temperature dependence of chromophore formation, and estimation and normalization of GFP levels.
To determine the quantitative limits of GFP in cells, several GFP versions (wtGFP, αGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F)) were compared by imaging of GFP expressing cells or by spectroscopic measurements of purified proteins. When imaged, the GFP signals are contaminated by the naturally occurring background autofluorescence, but improved detection can be achieved for each green GFP by combination of confocal microscopy using 488 nm excitation, a rapid cut-on dichroic mirror, and a narrow bandpass emission filter (Figure l).
GFP-Tagged Protein Domains as Tools to Study Localized Second Messenger Function in Living Vells
Authors: Meyer T , Oancea E , Stauffer T , Teruel M N 
Published: July 2020

Abstract
We have investigated the translocation of different GFP-tagged protein domains in response to various receptor stimuli. Confocal fluorescent imaging was used to study the localization of the GFP-tagged protein domain before, during and after the receptor stimuli was applied. A minimal cysteine-rich domain from protein kinase C was shown to be a useful tool to study localized diacylglycerol changes. Furthermore, the calcium-induced plasma membrane translocation of the GFP-tagged C2-domain from PKC could be used to study receptor-mediated changes in near plasma membrane calcium concentration. In addition, different pleckstrin homology domains (PH-domains) could be employed to measure changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI4.5P2) as well as of P13,4P2and PO,4,5P3. In combination, these fluorescent translocation probes provide a new means to investigate the local and temporal control of cell signaling processes in individual living cells.
Dynamin Mediates Membrane Vesiculation
Authors: Sweitzer Sharon M , Hinshaw Jenny E 
Published: July 2020

Abstract
Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis and synaptic vesicle recycling; however its mechanism of action is unknown. The requirement for dynamin was first elucidated by the discovery that the shibire gene product in Drosophila melanogaster was homologous to mammalian dynamin-1 (1,2). The shibire flies exhibit a depletion of synaptic vesicles and an accumulation of collared clathrin-coated pits at the plasma membrane of their nerve termini (3). It was later demonstrated that endocytosis was inhibited by the overexpression of dominant negative mutants of dynamin (4,5), and that purified dynamin can self-associate to form spirals which resemble the collars of shibire and structures seen in synaptosomes treated with GTPγS (6,7). These observations led to the speculation that dynamin pinches the clathrin-coated bud from the plasma membrane. In support of this hypothesis, we show that purified recombinant dynamin can bind to a lipid bilayer in a regular and repeating pattern to form helical tubes which vesiculate upon the addition of GTP.
Transport Through the Secretory Pathway: Observations of Cargo and Peripheral Coat Proteins
Authors: Presley John F , Cole Nelson B , Lippincott-Schwartz Jennifer 
Published: July 2020

Abstract
We have used green fluorescent protein (GFP) chimeras to examine the dynamics of the early secretory pathway and the role of the peripheral coat protein COP I. To describe the overall properties of ER to Golgi transport we used the temperature sensitive viral glycoprotein, ts045 VSVG, tagged with GFP at its cytoplasmic tail. VSVG-GFP retained the temperature sensitive phenotype of its parent: it reversibly misfolded and was retained in the ER at 40°C. Upon shift to 32°C it was rapidly exported from the ER, moving as a synchronous pool into the Golgi complex and then to the cell surface. Using time-lapse imaging of living cells expressing VSVG-GFP we found that the carriers for ER to Golgi traffic are tubulovesicular pre-Golgi intermediates that move centrosomally to the Golgi at speeds of&gt;1 μM2/sec and then fuse with the cis face of the Golgi complex. These movements are dependant on microtubules and the dynein/dynactin complex.
Neutrophil Microtubules Display Unusual Properties and are Highly Sensitive to Taxol
Authors: Robinson JM , Vandré DD 
Published: July 2020

Abstract
The microtubule (MT) cytoskeleton plays a crucial role in a number of cellular functions including cytoplasmic organization, intracellular transport, cellular polarity, and cell division. Microtubules are linear polymeric structures composed of heterodimers of α- and β-tubulin with a class of specialized proteins, the microtubule-associated proteins, typically being present. Microtubules are dynamic structures that undergo repeated rounds of assembly/disassembly. The half-life of MTs, an index of their dynamics (i.e., assembly-disassembly), has been estimated from studies of single cells in culture and typically ranges from ≈ 5 - 20 minutes depending on the cell type. These dynamic properties of MTs have been explained in terms of the “dynamic instability” model. Five parameters have been used to describe dynamic instability of MTs: (1) elongation velocity; (2) shortening velocity; (3) nucleation frequency; (4) catastrophe frequency; and (5) rescue frequency. Catastrophe is the transition from elongation to shortening while rescue is the transition from shortening to elongation.
Molecular Regulation of Taxane-Induced Apoptosis
Authors: Bhalla K 
Published: July 2020

Abstract
Following intracellular uptake, taxanes bind to β tubulin and suppress microtubular dynamics, resulting in stable microtubule bundles and mitotic arrest of cancer cells. This proximal taxane- β tubulin interaction depends on the amount of intracellular accumulation of taxanes. Since taxanes are substrates for the membrane transporter P-glycoprotein (PGP) but not the MRP, overexpression of PGP but not MRP reduces the intracellular accumulation and cytotoxicity of taxanes. Recent reports have also suggested that alterations in the levels of specific β tubulin isotypes can reduce the effects of taxanes on microtubule dynamics, thereby inhibiting the mitotic effects and resulting cytotoxicity of taxanes.6 For example, increased βIV and (βIII tubulin levels may confer resistance against microtubule and cytostatic effects of taxanes. Taxane-induced G2/M arrest is preceded by the activation of cell-cycle mitotic kinase CDK1, which promotes the G2/M transition. Recent studies have indicated that the loss of cell-cycle Gl checkpoint due to p53 mutations may enhance taxane-induced mitotic arrest and cytotoxicity.
Taxol-Induced Apoptosis May Occur Via a Signaling Pathway Independent of Microtubule Bundling and Cell Cycle Arrest
Authors: Fan Weimin , Miller III Merrill C , Cheng Lirong , Willingham Mark C 
Published: July 2020

Abstract
Taxol, a plant-derived antimicrotubule agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. This naturally occurring antineoplastic drug has been demonstrated to possess broad activity against a variety of human solid tumors, particularly in drug-refractory ovarian cancer and metastatic breast cancer (1). However, the exact mechanism of taxol's cytotoxicity against tumor cells is not entirely clear. Recent studies have demonstrated that taxol, besides causing microtubule bundling and mitotic arrest, is able to induce internucleosomal DNA fragmentation and typical morphological features of apoptosis in a number of solid tumor cells. These results clearly indicate that taxol, in addition to its classical activity against microtubules and cell cycle arrest, also possesses significant cell-killing activity by induction of apoptosis.
Although it is well recognized that taxol can cause both mitotic arrest and apoptotic cell death, it remains unclear whether taxol-induced cell death is a secondary event resulting from mitotic arrest or represents a novel mechanism of taxol against tumor cells.
Towards Understanding the Structural Requirements for Endogenous Transcription in Rotavirus
Authors: Lawton J A , Estes M K , Venkataram Prasad B V 
Published: July 2020

Abstract
Rotaviruses are complex, relatively large (1000 Å) nonenveloped icosahedral viruses. They are the major causative agents of severe diarrhea in children and animals. Annually an estimated 1 million children die from rotavirus-induced gastroenteritis worldwide. Medical relevance, intriguing structural complexity, and several unique strategies in the morphogenesis and replication have provoked extensive molecular biological and structural studies on rotaviruses in recent years.
Rotaviruses are double-stranded RNA (dsRNA) viruses belonging to the family Reoviridae. Each of the 11 segments codes for one protein. Of the eleven proteins encoded by the genomic RNA, six are structural and five are non-structural. Structural studies have shown that mature rotaviruses are triple-layered particles (TLPs). During the process of cell entry the outer-most layer consisting of VP7 and the spike protein VP4 is lost and the resulting double layered particles (DLPs) become transcriptionally active (Fig. 1 A). During the process of transcription, the DLPs maintain their structural Integrity.
The Application of Microscopy and Other Imaging Techniques to the Study of Disease in the Fetus.
Authors: Blackburn WR , Hudson J , Cooley,Jr NR 
Published: July 2020

Abstract
The application of both classical and advanced microscopy to the exploration of abnormal, human, fetal development (birth defects) and to other forms of human, fetal disease has generated a wealth of information, often documented by dramatic images. We have studied, at varying levels of inquiry (simple inspection to molecular analysis), some 10,000 normal and abnormal specimens. For presentation purposes, we first demonstrate the use of imaging in explorations of the fetal habitat (the gestational sac). Our studies reveal a variety of fetal surface encounter lesions (skin-to-amnion). Many of these lesions in the past have been erroneously interpreted. These include fetal amputations, strangulations, and also the reimplantation of amputated parts to ectopic sites. We have also explored, by various imaging techniques, developmental abnormalities within a variety of fetal organs.
A Morphometric Analysis of Early Effects of Ethanol on Hepatocyte Organelles from Peromyscusmaniculatus
Authors: Ellzey J T , Drake J P , Dader L , Boentges P 
Published: July 2020

Abstract
Pathological changes of hepatocytes from rats fed a 30% ethanol-derived calories diet for three weeks include noticeable ultrastructural changes including steatosis and hypertrophy of the smooth endoplasmic reticulum. We sought to examine hepatocytes of deer mice administered ethanol in an inhalation chamber for two weeks to determine if subtle changes occur in hepatocyte organelles prior to steatosis.
Two strains of Peromyscus maniculatus, ADH-positive possessing hepatic cytosolic alcohol dehydrogenase and ADH-negative deer mice lacking this enzyme were purchased from the Peromyscus Genetic Stock Center (Univ. of South Carolina). They tested negatively for Hanta viruses. A morphometric analysis of the ultrastructure of ADH+(n=14) and ADH- (n=14) controls as well as experimentals exposed to chronic, intoxicating levels of ethanol was conducted. Blood ethanol levels were maintained between 1.25-1.75 mg/ml for two weeks in the experimentals.
Sertoli Cell Adhesion Molecules: Comparative Expression of Lselectin and Other Cams in Primary Cells And Immortalized Sertoli Cell Lines
Authors: Broome Ann-Marie , Millette Clarke F 
Published: July 2020

Abstract
Cell adhesion and cell adhesion molecules (CAMs) play a crucial role in testicular development and function. The seminiferous epithelium, the functional unit of the testis, represents a three dimensional architecture of supporting Sertoli cells (SC), and developing germ cells (GC). The seminiferous epithelium, therefore, must be receptive not only to individual cell growth and differentiation, but also to cell-cell interactions. Morphologically distinct cell-cell interactions occur between SC and GC and also between SC.[1] In general, these junctions can be categorized into three types: adhesive, occluding, and gap junctions. The orientation and function of these junctions are interaction dependent. For example, desmosome-like junctions (spot desmosomes) are found between SC and GC. These junctions are present in the basal and intermediate compartments of the testis and serve to translocate developing GC. SC-SC interactions, like the zonula occludens (tight junction), function as vectorial mediators, maintaining the blood-testis barrier and SC polarity.
Nitric Oxide Synthase in Diabetic Retinopathy in the BBZ/WOR Rat: An Immunocytochemical Study
Authors: Ellis E Ann , Grant Maria B , Guberski Dennis L 
Published: July 2020

Abstract
Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and the highly reactive free radical species, NO. Two major constitutive isoforms, neuronal and endothelial NOS, have been ented in the retina. These constitutive isoforms produce small amounts of NO while the inducible isoform, iNOS, produces high levels of NO. Excess production of NO by iNOS has beem implicated in the complications of diabetes. Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS.
Semiquantitative colloidal gold labeled immunocytochemical studies of endothelial NOS (eNOS) and iNOS were done on sections of neural retina in eyes of obese, noninsulin dependent diabetic BBZ/Wor rats with diabetes of five months to twelve months duration. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.
Levels of iNOS were elevated in diabetic (33.9 ± 10.0 gold particles) as compared to age matched nondiabetic control (3.5 ± 2.8 gold particles) retinas.
The Effects of Cigarette Smoke on Porcine Airway Epithelium
Authors: Morgan Darrell D , Moss Anthony G 
Published: July 2020

Abstract
Cephalad pulmonary mucociliary clearance driven by cilia of the ciliated airway epithelium provides probably the most important line of defense against inhaled toxins and particulate material for organs of the thoracic cavity. We demonstrate the reorganization of the cytoskeleton and the endoplasmic reticulum of airway epithelial cells upon cigarette smoke inhalation by employing DIC, LSCM, TEM and widefield fluorescence microscopy and have correlated this reorganization to changes in ciliary beat frequency (CBF) via FFT analysis. Neonatal pigs were used to provided healthy tracheal epithelial tissue. Exposure to cigarette smoke causes rapid ultrastructural changes including: ciliary distortion and detachment, ciliary abscission and severe alteration in endoplasmic reticulum structure suggesting profound disruption of essential membrane-cytoskeletal linkages.
To examine changes in CBF we describe a simple approach, using a laser scanning confocal microscope (LSCM) and an analog-to-digital computer converter/analyzer for the acquisition of data from biological systems that undergo rapid periodic movement, such as ciliary motion.
ION-Channel Activator-Induced Ultrastructural Changes in the Dog Retina
Authors: Megill JR , Monticello TM , Wasserman AJ , Kelly WA , Durham SK 
Published: July 2020

Abstract
The sensory portion of the retina is composed of multiple cell layers with complex functions and interactions vital to ocular homeostasis. Xenobiotics can induce physiological and morphological perturbations of these cell layers, and subsequently compromise vision. We describe an ion channel activator (ICA)-induced retinopathy observed in a preclinical drug safety study in dogs. Purebred beagle dogs received either ICA in vehicle (0.75% Methocel® and 0.05% Tween 80) or vehicle alone (control) for nine months. During the study, visual deficits and fundic changes were observed clinically, leading to premature termination of the study at eight months. The posterior globe of the left eye was fixed by the method of Acland and Aguirre, dehydrated in a series of graded methanol baths, and embedded in epoxy resin. Semi-thin (l-μm thick) sections of the retina were stained with toluidine blue, and examined by light microscopy. Ultrathin (60-90-nm thick) sections of the retina were mounted on copper grids,
Electron Microscopic Immunolocalization of Basic Fibroblast Growth Factor-Like Molecules in Capillary Endothelial Cells
Authors: Aktas Ranan Gullhan , Kayton Robert J 
Published: July 2020

Abstract
Basic fibroblast growth factor (bFGF) is a potent angiogenic polypeptide. It promotes angiogenesis in vivo and in vitro by stimulating migration, proliferation and proteolytic activity of endothelial cells. Whereas several effects of exogenous bFGF on endothelial cells have been described, it has remained unclear how endogenous bFGF produced by vascular endothelial cells regulate angiogenesis.
To further investigate functional implications of the distribution of bFGF, we undertook the present study. Our aims were (i) to identify the specific location of bFGF in endothelial cells using electron microscopy immunogold labeling technique (ii) to determine the distribution of bFGF in capillaries of different types of tissues.
Tissue samples from sciatic nerve, hippocampus, adrenal gland and kidney of normal adult rats were fixed in 4% paraformaldehyde/1 to 5% glutaraldehyde and embedded in Spurr's resin. Ultrathin sections were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics) specific for bFGF using a two-step immunogold labeling method.
Acid Phosphatase Activity in Spiral Ganglion Neurons of C57BL/6 Mice
Authors: Cohen Glenn M 
Published: July 2020

Abstract
C57BL/6 mice, along with several other mouse genotypes, have served as models for human presbycusis (age-related hearing losses). C57BL/6 mice and their genetic substrain C57/M6 show progressively severe hearing losses, starting as early as 30 days postnatally. The hearing losses result from sweeping degeneration of sensory (hair) cells and neurons that begins in the basal end of the cochlea and advances apically. Although the underlying mechanisms orchestrating sensory and neural degeneration are not known, it is possible to correlate degenerative events with the cytoplasmic levels and distribution patterns of a marker molecule, such as acid phosphatase (AP). AP, a representative lysosomal enzyme, plays a role in both normal cellular metabolism and degenerative changes (trauma and senescence). AP activity is visualized histochemically at the light and electron microscopic levels by the presence of dense deposits within lysosomes.
Age- and Condition-Related Changes in Levels of Neuron Specific Enolase in Spiral Ganglion Neurons of C57BL/6 Mice
Authors: Cohen Glenn M 
Published: July 2020

Abstract
The mouse strain C57BL/6 suffers from the early onset of deafness. The deafness is attributable to sweeping degeneration of hair cells and the auditory nerve. Degeneration advances with age from the base to the apex of the cochlea. In the present study, neuronal condition was determined by linking relative levels of neuron specific enolase (NSE) in spiral ganglion neurons to their cytological appearances. NSE is an enolase isoform that is expressed only in nervous tissue and neuroendocrine glands.
Mice ranged in age from 3 days to 10 months. Inner ears were fixed with 2.5% paraformaldehyde in 0.1 M cacodylate buffer, decalcified for 24 hrs with EDTA, and then embedded in either paraffin or polyester wax. The latter, which melts at a lower temperature, caused much less neuronal shrinkage during routine processing. Deparaffinized sections were exposed first to rabbit anti-bovine NSE antibodies, then to biotinylated swine anti-rabbit antibodies, and finally to peroxidase-labelled avidin.