A Golgi deimpregnation study of neurons in the rhesus monkey visual cortex (Areas 17 and 18)

Summary The morhological features of 298 neurons impregnated according to Golgi-Kopsch in areas 17 and 18 of Macaca mulatta were analyzed, and the same neurons were deimpregnated to visualize structural details of the somata in different types of neurons. The following cell types were investigated: Pyramidal and pyramid-like cells, spiny stellate cells, double bouquet cells, bipolar cells, chandelier cells, neurogliaform cells, basket and related cells. This procedure allows the evaluation of the nuclear-cytoplasmic proportion and the position of the nucleus besides shape and size of the cell body. Pyramidal and pyramid-like cells (N=43), spiny stellate cells (N=26), basket and related cells (N=126) are variable in these features. A positive correlation between soma size and width of the cytoplasm is found in pyramidal, pyramid-like cells and spiny stellate cells. With the exception of some large somata in both these types of neurons the nucleus is found in a central position. Double bouquet cells (N=6), bipolar cells (N=13) and chandelier cells (N=11) exhibit small cytoplasmic rims and centrally located nuclei. The small somata of neurogliaform cells (N=37), however, and the small to very large somata of basket and related cells show broad cytoplasmic portions surrounding the eccentrically located nuclei. These findings allow the identification of different neuronal types in Nisslstained sections on the basis of these soma features. This is a prerequisite for further detailed quantitative studies on the laminar distribution of different neuronal types in the visual cortex of the monkey.     

Relationship between angiographic late loss and target lesion revascularization after coronary stent implantation: analysis from the TAXUS-IV trial

OBJECTIVES: We sought to evaluate the relationship between angiographic late loss and clinical outcomes in the drug-eluting stent era. BACKGROUND: The interrelationship between angiographic late loss, binary restenosis, and clinical recurrence (target lesion revascularization [TLR]) after coronary stent implantation has been incompletely evaluated. METHODS: Using the angiographic substudy of the TAXUS-IV trial, in which 1,314 patients with de novo coronary lesions were randomized to either the paclitaxel-eluting TAXUS stent or to its bare-metal equivalent, we defined the relationship between in-stent and analysis segment late loss, the shape of the late loss histogram (variance and skewedness), and nine-month TLR. RESULTS: Late loss by several measures was closely related to TLR (area under the receiver-operator curve >0.90). For individual vessels of the size in this study (2.8 +/- 0.5 mm), the likelihood of TLR did not exceed 5% until analysis segment late loss was >0.5 mm, and did not exceed 10% until late loss was >0.65 mm. At greater late losses, the late loss TLR relationship was steep and nearly linear. For the overall patient cohort, the rate of TLR was related, however, not only to median late loss, but also to measures of its statistical distribution (TLR increased with lack of homogeneous biologic response [greater variance and greater right skewedness]). Similar relationships held for late loss measured within the confines of the stent itself. CONCLUSIONS: Coronary stents result in large lumens with "room" to accommodate up to approximately 0.5 to 0.65 mm of tissue (angiographic analysis segment late loss) before the likelihood of clinical restenosis (TLR) exceeds 5% to 10%. These data have important implications toward understanding the absolute and relative efficacy of drug-eluting stents.     

Protein crystal structure obtained at 2.9 ? resolution from injecting bacterial cells into an X-ray free-electron laser beam

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.The advent of X-ray free-electron lasers (XFELs) has made it possible to obtain atomic resolution macromolecular structures from crystals with sizes approximating only 1/60th of the volume of a single red blood cell. Brief, intense pulses of coherent X-rays, focused on a spot of 3-μm diameter, have produced 1.9-Å-resolution diffraction data from a stream of lysozyme crystals, each crystal no bigger than 3 μm³ (1). A stream of crystals, not just one crystal, is required to collect the many tens of thousands of diffraction patterns that compose a complete data set. No single crystal can contribute more than one diffraction pattern because the XFEL beam is so intense and the crystals so small that the crystals are typically vaporized after a single pulse. Impressively, a photosystem I crystal no bigger than 10 unit cells (300 nm) on an edge produced observable subsidiary diffraction peaks between Bragg reflections, details which would be unobservable from conventionally sized crystals (2). With this new ability to collect diffraction patterns from crystals of unprecedentedly small dimensions, it is conceivable that high-resolution diffraction data could be collected from crystals in vivo. The structure obtained in this manner would be unaltered from that occurring naturally in a living cell, free from distortion that might otherwise potentially arise from nonphysiological conditions imposed by recrystallization. A practical advantage would also be gained by eliminating the need for a protein purification step, whether the in vivo grown crystals were naturally, or heterologously expressed (3).The nascent field of serial femtosecond crystallography (SFX) has published results on nine different macromolecular systems since its inception in 2009 (3, 9). The crystals for this study were not grown in artificial crystallization chambers as has been the protocol of conventional macromolecular crystallography since the 1950s. Instead, crystals were grown in cells. Specifically, they were grown in Sf9 insect cells, heterologously expressing Trypanosoma brucei cathepsin B. These in vivo-grown crystals were used for the XFEL diffraction experiment. To this end, the cells were lysed and the crystals were extracted before injecting them in the XFEL beam for data collection. This last purification step seems to be the only major departure from our goal of obtaining high-resolution structural information from crystal inclusions in vivo, without requiring the crystal to be extracted from the cell that assembled it. Here we attempt to go one step further than previous studies—to record diffraction from crystals within living cells.

Table 1.

SFX publications from XFEL sources to date

Biliverdin IX is an endogenous inhibitor of soluble guanylyl cyclase

Heme oxygenase (HO) converts heme to carbon monoxide (CO) and biliverdin IX. CO is a weak activator of soluble guanylyl cyclase (SGC), the enzyme that catalyzes the conversion of GTP to the second messenger cGMP. HO overexpression has recently been shown to inhibit production of cGMP by SGC in vivo. The aim of the present study was to investigate a possible influence of biliverdin IX on SGC activity. Using recombinant alpha(1)/beta(1) isoform of SGC, we show an inhibitory effect of biliverdin IX in the micromolar range both on basal and NO stimulated guanylyl cyclase activity. Bilirubin IX which differs from biliverdin IX in two hydrogen atoms had no effect. Biliverdin IX reduced maximal guanylyl cyclase activity (V(max) values) while it had no effect on the K(M) values indicating unchanged affinity towards the substrate GTP. Concentration response experiments using the NO donor, 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO), showed that enzyme activities at maximal DEA/NO concentration were reduced by biliverdin IX. The affinity of the NO-donor, DEA/NO, towards SGC was significantly reduced in the presence of biliverdin IX. Biliverdin IX lowered enzyme activity at maximal activator concentrations of YC-1 and protoporphyrin IX (PPIX) while it had no significant effect on the EC(50) values of these two NO independent activators. The inhibitory effect of biliverdin IX on PPIX activated enzyme activity is not shared by ODQ, which indicates that the inhibitory mechanism of biliverdin IX is different from ODQ.     

Utility of [<Superscript>18</Superscript>F]FSPG PET to Image Hepatocellular Carcinoma: First Clinical Evaluation in a US Population

Purpose

Non-invasive imaging is central to hepatocellular carcinoma (HCC) diagnosis; however, conventional modalities are limited by smaller tumors and other chronic diseases that are often present in patients with HCC, such as cirrhosis. This pilot study evaluated the feasibility of (4S)-4-(3-[¹⁸F]fluoropropyl)-L-glutamic acid ([¹⁸F]FSPG) positron emission tomography (PET)/X-ray computed tomography (CT) to image HCC. [¹⁸F]FSPG PET/CT was compared to standard-of-care (SOC) magnetic resonance imaging (MRI) and CT, and [¹¹C]acetate PET/CT, commonly used in this setting. We report the largest cohort of HCC patients imaged to date with [¹⁸F]FSPG PET/CT and present the first comparison to [¹¹C]acetate PET/CT and SOC imaging. This study represents the first in a US HCC population, which is distinguished by different underlying comorbidities than non-US populations.

Procedures

x_C? transporter RNA and protein levels were evaluated in HCC and matched liver samples from The Cancer Genome Atlas (n = 16) and a tissue microarray (n = 83). Eleven HCC patients who underwent prior MRI or CT scans were imaged by [¹⁸F]FSPG PET/CT, with seven patients also imaged with [¹¹C]acetate PET/CT.

Results

x_C? transporter RNA and protein levels were elevated in HCC samples compared to background liver. Over 50 % of low-grade HCCs and ~70 % of high-grade tumors exceeded background liver protein expression. [¹⁸F]FSPG PET/CT demonstrated a detection rate of 75 %. [¹⁸F]FSPG PET/CT also identified an HCC devoid of typical MRI enhancement pattern. Patients scanned with [¹⁸F]FSPG and [¹¹C]acetate PET/CT exhibited a 90 and 70 % detection rate, respectively. In dually positive tumors, [¹⁸F]FSPG accumulation consistently resulted in significantly greater tumor-to-liver background ratios compared with [¹¹C]acetate PET/CT.

Conclusions

[¹⁸F]FSPG PET/CT is a promising modality for HCC imaging, and larger studies are warranted to examine [¹⁸F]FSPG PET/CT impact on diagnosis and management of HCC. [¹⁸F]FSPG PET/CT may also be useful for phenotyping HCC tumor metabolism as part of precision cancer medicine.

     

Classification of neurons in the visual cortex of the guinea pig (Cavia porcellus). A combined Golgi-Nissl study using deimpregnation technics

The aim of the investigation is the identification of neuronal types in the visual cortex of the guinea pig (Cavia porcellus) in Nissl preparations. In two rapid Golgi series (modified by Fairén et al. 1977) and nine Golgi-Kopsch series eleven neuronal types could be classified: 1. Neurons with long axonal main stems: Pyramidal cells (layers II-VI), pyramid-like cells (layers I, VI), spiny stellate cells (layer IV). 2. Neurons with locally distributed axons: large and small aspiny cells (layers II-VI), neuroglioform cells (layers I-VI), large sparsely spiny cells with ascending axons (Martinotti cells) (layers III-VI), small sparsely spiny cells with variable axons (layer IV), bipolar cells (layers II-VI), chandelier cells (layers II, III), double bouquet cells (layers II, III), and 3. a horizontal cell in layer I without an impregnated axon. To identify Nissl stained somata, Golgi impregnated neurons were deimpregnated. Now cytoplasmic and nuclear features can be compared. In order to get a survey about the variability of the somal features, we have deimpregnated 245 neurons on the whole using either gold chloride (Fairén et al. 1977) combined with the rapid Golgi method, modified by Fairén et al. (1977), or a diluted solution of ammonia (Braak and Braak 1982) combined with the Golgi-Kopsch method, nonembedded, in glycerol stored material and in celloidin embedded sections, resp. After deimpregnation several neuronal classes could be distinguished considering at first the width of the cytoplasm and the localization of the nucleus. There are Nissl stained somata with corresponding features: 1. Pyramidal cells, pyramid-like cells, and spiny stellate cells of the layer IV with relatively broad cytoplasm and bright, centrally localized nuclei. 2. Large aspiny cells which vary in somal sizes and shapes with very broad cytoplasm and bright, centrally or excentrally localized nuclei. 3. Small aspiny cells, some of the neuroglioform cells, and small sparsely spiny cells of layer IV have more or less broad cytoplasm but in the most cases excentrally localized nuclei. 4. The remaining neuroglioform cells, bipolar cells, and the horizontal cell of layer I are particularly poor in cytoplasm. The somata of these neuronal types are small, those of the bipolar cells are vertically orientated. 5. Like the bipolar cells large sparsely spiny cells with ascending axons Martinotti cells), chandelier cells, and double bouquet cells are also polarized neurons, but larger and not always vertically oriented.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-gamma knockout recipients.

To investigate the functional role of interferon (IFN)-gamma in transplant arteriosclerosis, BALB/c hearts were transplanted in immunosuppressed C57BL/6J recipients with (n = 10) or without (n = 10) targeted IFN-gamma gene deletion. In 55-day heart allografts, IFN-gamma deficiency resulted in a significant decrease in vascular thickening. The severity of intimal thickening measured as the percentage of luminal occlusion (mean +/- SEM) in all elastin stained vessels (n = 410) decreased from 37+/-5% in wild-type recipients to 18+/-5% in IFN-gamma -/- recipients (P < 0.005). In the few diseased vessels in grafts from IFN-gamma -/- recipients, the neointima was more cellular with a 90% increase in the nuclear density. This finding correlated with a 50% reduction in fibrosis estimated by alpha-smooth muscle actin cell accumulation in the neointima. The reduction in severity and altered composition of vascular thickening in grafts from IFN-gamma -/- recipients shows that IFN-gamma contributes to arteriosclerotic development following transplantation.