Chromatin pattern by variogram analysis.

Many cytological processes such as cell proliferation, differentiation, transformation, apoptosis, etc., are accompanied by specific chromatin changes, usually identified on the basis of the relative content of euchromatin and heterochromatin. In order to achieve a quantitative, non-subjective evaluation of the chromatin pattern, two different approaches may be undertaken, one consisting in the analysis of the several morphological features of chromatin grains (size, shape, density, arrangement, and distribution), and the second consisting in the analysis of the chromatin globally considered as a coherent texture. Although the second approach appears to be simpler and more suitable, methods of texture analysis--including those specifically designed for the analysis of the chromatin pattern--are rarely applied due mainly to the unsuitability of sampling procedures and the excessive crypticism of results. As an alternative to traditional texture analysis, we suggest a method supported by a sound mathematical theory and approximately 30 years of applications in the field of geostatistics. The method, called variogram, analyzes the intrinsic structure of data sampled at different distance intervals and directions, and outputs easily understandable results. Recently, variogram analysis has successfully been exported from geostatistics to other fields (for example, ecology and epidemiology) that make use of spatially referenced variables. Based on the fact that pixels represent a perfect array of data ordered at regular distance intervals and directions, the variogram can be adopted to explore nuclear images and recognize chromatin patterns. Variograms of different nuclei can be summarized by multivariate methods without the need of previous standardization of data. This allows comparison and discrimination of chromatin patterns from mixed cell populations. Preliminary data obtained from young neurons undergoing massive apoptosis reveal a self-consistent map of nuclear changes correlated to the degenerative process.

trk-like immunoreactivity in the human trigeminal ganglion and subnucleus caudalis.

The immunohistochemical occurrence of trkA, trkB and trkC receptors was examined in the human trigeminal ganglion and spinal nucleus of subjects at all ages and compared with that of substance P (SP) and calcitonin gene-related peptide (CGRP), trk-like immunoreactive (LI) material was detectable in discrete subpopulations of primary sensory neurones from 25 weeks of gestation to adult life. Each subpopulation overlapped partially with those immunoreactive to SP and CGRP, trkA- and trkC-positive filamentous and punctate elements occurred in the trigeminal subnucleus caudalis. While immunostaining for trkC was restricted to rare isolated elements, that for trkA outlined the superficial laminae of the nucleus and was more intense early in life than in adults.

GAP-43 persists in adulthood and coexists with SP and CGRP in human trigeminal sensory neurones.

Immunohistochemistry was used to examine the occurrence and colocalization of the growth-associated protein GAP-43 with substance P (SP) and calcitonin gene-related peptide (CGRP) in the human trigeminal ganglion and sensory nucleus at perinatal and adult life stages. The results obtained show that: GAP-43-like immunoreactive (LI) material persists in trigeminal primary sensory neurones of the normal adult; the GAP-43-LI ganglionic population partially overlaps with those immunoreactive to SP and CGRP; the distribution pattern of the protein in the spinal nucleus varies with age; in the adult subnucleus caudalis GAP-43 is co-distributed with SP and CGRP. It is suggested that the trigeminal GAP-43-LI neuronal system may retain the capacity for structural and functional plasticity in adult life.

Nuclear pattern recognition by two-parameter texture analysis.

The present paper describes a simple procedure for the analysis of chromatin texture. High-resolution digitized images of nuclei are first standardized to render gray values invariant to staining and illumination conditions. Subsequently, the nucleus is subdivided by a square grid into 0.4 x 0.4 microns2 quadrats and standard deviations of gray values within each quadrat are estimated. Finally, the overall mean and standard deviation of quadrat standard deviations are calculated. These values may be considered as pure descriptors of the nuclear texture, as they represent the distribution of chromatin changes, disregarding any absolute densitometric and morphometric feature. Using the above descriptors it is possible to recognize at least seven chromatin patterns in a mixed population of developing and degenerating neurons. Results are visually verified by mapping the original pictures at the corresponding bivariate plot points. Comparison with the Markovian texture analysis is discussed.

The efficiency of in-situ hybridization on human chromosomes with alphoid DNAs is enhanced by previous digestion with AluI and TaqI.

Centromeric alphoid DNAs of human chromosomes 6, 9, 16 and Y were employed to obtain information on the molecular mechanism(s) determining cytological effects produced by digestion in situ with AluI and TaqI restriction enzymes, possibly related to the structure of the above-cited areas. The following cytological and biochemical experiments were carried out using the above-mentioned alphoid sequences as probes: (1) standard in-situ hybridization and in-situ hybridization after chromosome cleavage with AluI/TaqI, and (2) filter hybridization on the DNA fractions obtained from the material solubilized and that retained on the slides after digestion in situ with AluI/TaqI. Biochemical data show that cleavage of alphoid DNAs is not prevented by the peculiar organization of centromeric heterochromatin, but such cleavage is not necessarily followed by complete DNA solubilization. The analysis of alphoid sequence cleavage in naked genomic DNA as well as during digestion of fixed chromosomes shows that (1) AluI cuts more efficiently than TaqI, (2) DNA fragments as large as 3-5 kb can be solubilized, and (3) DNA fragments of the same size are found in both fractions of DNA, i.e. that retained on the chromosomes as well as that solubilized from chromosomes. Cytological data show that previous chromosome digestion, mostly with TaqI, increases the hybridization signal area, suggesting that this fact might be due to (1) chromatin reorganization produced by enzyme attack and/or (2) the presence of alphoid DNAs which might be restricted not only to the kinetochore area but also to para/peri-centromeric heterochromatin. Lastly, centromere DNA solubilization as a consequence of restriction enzyme cleavage seems to vary from chromosome to chromosome, thus suggesting that centromeric regions do not represent a homogeneous class of constitutive heterochromatin.

Subcellular heterogeneity of mitochondrial membrane potential: relationship with organelle distribution and intercellular contacts in normal, hypoxic and apoptotic cells.

The subcellular heterogeneity of mitochondrial membrane potential (mDelta psi) was investigated in confluent and sub-confluent cultures of four cell types (human astrocytes, HEp-2, MDCK and Vero cells) in normal growth conditions, hypoxia and apoptosis. The distribution of high-polarized mitochondria, detected by the potential-sensitive probe JC-1, was found to depend on: (1) the proximity to the cell edge; (2) the local absence of cell-cell contacts; and (3) the local absence of acidic vesicles. Both hypoxia and apoptosis produced a general mDelta psi increase with different redistributions of high-polarized mitochondria. Hypoxic cells maintained high-polarized mitochondria for over 24 hours, until cells underwent necrosis. On the other hand, apoptotic cells showed an unexpected convergence of high-polarized mitochondria into an extremely packed mass at one side of the nucleus, in a stage preceding nuclear condensation, but correlated to the retraction of cell-cell contacts.