Towards a quantitative grading of bladder tumors.

The histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated. Three hundred thirty-three images were digitized from 111 bladder tissue sections (4 microns thick, Feulgen stained), using the ICAS microscope-camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity. The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as "in between grade 1 and grade 3" and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population. The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields-of-view are chosen for analysis.     

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Antibody responses of mice exposed to low-power microwaves under combined, pulse-and-amplitude modulation

Irradiation by pulsed microwaves (9.4 GHz, 1 microsecond pulses at 1,000/s), both with and without concurrent amplitude modulation (AM) by a sinusoid at discrete frequencies between 14 and 41 MHz, was assessed for effects on the immune system of Balb/C mice. The mice were immunized either by sheep red blood cells (SRBC) or by glutaric-anhydride conjugated bovine serum albumin (GA-BSA), then exposed to the microwaves at a low rms power density (30 microW/cm2; whole-body-averaged SAR approximately 0.015 W/kg). Sham exposure or microwave irradiation took place during each of five contiguous days, 10 h/day. The antibody response was evaluated by the plaque-forming cell assay (SRBC experiment) or by the titration of IgM and IgG antibodies (GA-BSA experiment). In the absence of AM, the pulsed field did not greatly alter immune responsiveness. In contrast, exposure to the field under the combined-modulation condition resulted in significant, AM-frequency-dependent augmentation or weakening of immune responses.     

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.     

Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha.

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.     

Glutamic acid decarboxylase of embryonic avian retina cells in culture: Regulation byγ-aminobutyric acid (GABA)

1. Retina-cell aggregate cultures expressed glutamate decarboxylase activity (L-glutamate 1-carboxylase; EC 4.1.1.15) as a function of culture differentiation. 2. Glutamic acid decarboxylase (GAD) activity was low in the initial phases of culture and increased eight-fold until culture day 7, remaining high up to day 13 (last stage studied). 3. The addition of GABA to the culture medium 24 h after cell seeding almost totally prevented the expression of GAD activity. 4. In association with decreased enzyme activity, aggregates exposed to GABA did not display immunoreactivity for GAD, suggesting that GAD molecules were either lost from GABAergic neurons or significantly altered with GABA treatment. 5. Control, untreated aggregates showed intense GAD immunoreactivity in neurons. Positive cell bodies were characterized by a thin rim of labeled cytoplasm with thickest labeling at the emergence of the main neurite. 6. Heavily labeled patches were also observed throughout the aggregates, possibly reflecting regions enriched in neurites. 7. The GABA-mediated reduction of GAD immunoreactivity was a reversible phenomenon and could be prevented by picrotoxin.