Hexokinase ‘binding’ of normal and tumoral human brain mitochondria

Interaction of type I hexokinase (HK-I) with the mitochondria obtained from the biopsy specimens of normal and tumoral human brain tissues was studied in the present investigation. This effort was undertaken with the aim of exploring possible differences in the mode of association of the enzyme with the outer mitochondrial membrane in the described sources. Results indicate that the two sites for binding of HK-I suggested in the literature, based on extensive studies carried out on rat brain mitochondria, are similarly present in the human brain mitochondria. Differences in the microenvironments of HK binding, as reflected by the presented data, are suggested to be of importance in regulation of the catalytic potential of the bound enzyme. The real metabolic significance of this association in relation to cancer and its practical importance would need further investigation.     

‘Conservative’ and ‘variable’ clusters of Alu-family DNA repeats in human chromosomes

The distribution of Alu-family DNA repeats (AFRs) in chromosomes of phytohaemagglutinin-stimulated peripheral blood lymphocytes of four normal donors and non-stimulated bone marrow cells of four patients with acute leukemia (ALL and ANLL) was studied by in situ hybridization using DNA of recombinant phage lambda containing multiple inserts of AFR as a probe. Over some chromosome bands (14cen, 16p13, 16cen) from normal donors and from leukemic patients clusters of silver grains were detected. Over other three bands (3q26, 8p11-p12 and 14q24) the clusters were found only in chromosomes from the four acute leukemia patients, and were absent from chromosomes of healthy donors. The results suggest non-random long-range distribution of AFRs in human chromosomes, and somatic variations in the distribution of the repeats.     

Strasburger’s legacy to mitosis and cytokinesis and its relevance for the Cell Theory

Eduard Strasburger was one of the most prominent biologists contributing to the development of the Cell Theory during the nineteenth century. His major contribution related to the characterization of mitosis and cytokinesis and especially to the discovery of the discrete stages of mitosis, which he termed prophase, metaphase and anaphase. Besides his observations on uninucleate plant and animal cells, he also investigated division processes in multinucleate cells. Here, he emphasised the independent nature of mitosis and cytokinesis. We discuss these issues from the perspective of new discoveries in the field of cell division and conclude that Strasburger's legacy will in the future lead to a reformulation of the Cell Theory and that this will accommodate the independent and primary nature of the nucleus, together with its complement of perinuclear microtubules, for the organisation of the eukaryotic cell.     

A comparative ultrastructural study of ‘glue’ production and secretion of the salivary glands in different species of theDrosophila melanogaster group

Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as glue at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.     

Immunofluorescence and ultrastructural localization of β2-microglobulin in human renal allografts

Summary The localization of 2-microglobulin (2m) was studied in renal biopsies from 18 patients with pathological transplanted kidney using immunofluorescence and electron-immunohistochemical techniques; the renal biopsies of 4 cases with normal kidneys were used as controls. Using immunofluorescence, 2m was not observed in the normal kidneys, 2m was found in the glomeruli (7 cases) and the tubular epithelium (16 cases) of the transplanted kidneys. Using immunoelectron microscopy, some labelling of the normal kidneys was observed mainly along the cell coat of foot processes and in tubular-epithelial lysosomes. In the glomeruli of transplanted kidneys, particularly in cases of acute or chronic rejection, 2m was most frequently localized on the outer layer of the basement membrane and along the cell coat of foot processes. The brush border of the proximal tubules and the lysosomal structures were intensely labelled. Although immunoelectron-microscopy studies are unable to discriminate between the localization of 2m in normal and transplanted kidneys, these findings nevertheless suggest the glomerular filtration of 2m and its metabolism in the tubular epithelium.     

The distribution of selenium in human blood samples of Israeli population—Comparison between normal and breast cancer cases

A preliminary study was carried out in order to compare the selenium concentration in breast cancer patients and healthy subjects (controls) in Israel. Blood serum samples were obtained from 32 breast cancer patients and 36 controls and were analyzed for selenium by the XRF method. A weighted mean of 0.076±0.014 ppm Se in the blood serum of breast cancer patients, as compared to 0.119±0.023 ppm Se for controls, was obtained. These results indicate that the concentration of selenium in breast cancer patients is significantly lower than in controls. The relationship between selenium concentration and malignancy stage shows an inverse dependence, i.e., the concentration decreases with stage number.     

An analysis of a ‘community-driven’ reconstruction of the human metabolic network

Following a strategy similar to that used in baker’s yeast (Herrgård et al. Nat Biotechnol 26:1155–1160, 2008). A consensus yeast metabolic network obtained from a community approach to systems biology (Herrgård et al. 2008; Dobson et al. BMC Syst Biol 4:145, 2010). Further developments towards a genome-scale metabolic model of yeast (Dobson et al. 2010; Heavner et al. BMC Syst Biol 6:55, 2012). Yeast 5—an expanded reconstruction of the Saccharomyces cerevisiae metabolic network (Heavner et al. 2012) and in Salmonella typhimurium (Thiele et al. BMC Syst Biol 5:8, 2011). A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella typhimurium LT2 (Thiele et al. 2011), a recent paper (Thiele et al. Nat Biotechnol 31:419–425, 2013). A community-driven global reconstruction of human metabolism (Thiele et al. 2013) described a much improved ‘community consensus’ reconstruction of the human metabolic network, called Recon 2, and the authors (that include the present ones) have made it freely available via a database at http://humanmetabolism.org/ and in SBML format at Biomodels (http://identifiers.org/biomodels.db/MODEL1109130000). This short analysis summarises the main findings, and suggests some approaches that will be able to exploit the availability of this model to advantage.     

Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit

Summary Human and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to cardiac , slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle cardiac myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express cardiac myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of cardiac myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The cardiac myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.     

Cell division study in ‘pollen mother cells’ and ‘pollen grains’ of in vivo and ex vitro plants in Drimiopsis botryoides

The various normal and abnormal stages of meiosis and pollen mitosis of Drimiopsis botryoides are described, and a comparison between naturally propagated in vivo and tissue culture derived ex vitro plants in respect to their cytological behaviour presented. We also describe the floral morphology and investigate the relationship between the floral developmental stages and the progression of microgametogenesis. In total, 33 bivalents are observed in diakinesis, which indicate the diploid number 2n = 66 and this number is cross-checked by a haploid set of n = 33 chromosomes in pollen mitosis. Only 6.8% and 4.9% meiotic abnormalities were recorded on in vivo and ex vitro plants, respectively, which led to the formation of non-viable pollen. Finally, the microspores have to develop into tri-cellular male gametophyte. Only 0.2% pollen grains are found with a micro-nucleus. Though the higher pollen viability was recorded on both in vivo (89.3 ± 4.1%) and ex vitro (92.1 ± 4.6%) plants, but surprisingly the pollen germination rate is extremely low with 13.6 ± 1.74% and 21.3 ± 1.55%, respectively. The present study obviously enriches the cytological database of D. botryoides and may help future research on androgenesis and genetic improvement.     

An explication of ‘wind illness’ in Northern Thailand

Wind illness is a very common complaint among the Northern Thai, yet is rarely recognized by Thai physicians trained in biomedicine. Persons most susceptible to wind illness are adult women who have ever borne a child. Consequently, data were obtained from 415 everparous women, 43% of whom reported ever having had wind illness and 57%, never having had it. In addition, 20 individuals who had ever had the syndrome were followed for case study, and 13 indigenous healers who traditionally treat clients suffering wind illness were interviewed. Their perceptions of the etiology, symptomatology and treatment of wind illness are reported in Part I. Part II is an attempt to define wind illness in terms of biomedicine and as a consequence of fertility. Part III synthesizes the emic and etic accounts with explanations for the perdurance of wind illness despite the advances of biomedicine and the recent fertility decline in Northern Thailand.     

Expression of class III β-tubulin in normal and neoplastic human tissues

 The class III β-tubulin isotype is widely used as a neuronal marker in normal and neoplastic tissues. This isotype was, however, also immunodetected in certain tumours of non-neuronal origin such as squamous cell carcinoma. Using a newly described monoclonal antibody we compared the distribution of class III β-tubulin in normal and neoplastic tissues. The TU-20 mouse monoclonal antibody was prepared against a conserved synthetic peptide from the C-terminus of the human class III β-tubulin isotype, and its specificity was confirmed by immunoblotting, by competitive enzyme-linked immunosorbent assay and by immunofluorescence microscopy on cultured cells. In different cell lines of various origins the antibody reacted only with neuroblastoma Neuro-2a cells and with embryonal carcinoma P19 cells stimulated to neuronal differentiation by retinoic acid. Immunohistochemistry on formaldehyde-fixed paraffin-embedded normal human tissues revealed the presence of the class III β-tubulin isotype in cell bodies and processes of neuronal cells in the peripheral and central nervous systems. In other tissues, this β-tubulin isotype was not immunodetected. Class III β-tubulin was found in all cases of ganglioneuroblastoma, ganglioneuroma, medulloblastoma, neuroblastoma, sympathoblastoma and in one case of teratoma. In contrast, no reactivity was detected in tumours of non-neuronal origin, including 32 cases of squamous cell carcinoma. The results indicate a specific TU-20 epitope expression exclusively in neuronal tissues. The antibody could thus be a useful tool for the probing of class III β-tubulin functions in neurons as well as for immunohistochemical characterisation of tumours of neuronal origin. Accepted: 29 July 1997     

Methylation of progesterone receptor isoform A promoter in normal breast tissue: An epigenetic link between early age at menarche and risk of breast cancer?

Emerging evidence indicates that some altered patterns of methylation that occur in breast tumors may also be found in breast tissue of healthy women in relation to the breast cancer (BC) risk factors. Progesterone receptor (PR) isoform α is a crucial regulator of breast hormone responsiveness and its hypermethylation plays an important role in the initiation and development of breast tumors. However, such a methylation change in healthy women and its link with the different risk factors has not yet been investigated. In the present study, we aimed to examine the relationship of possible methylation changes within a critical region in the promoter CpG island of PGR-α (progesterone receptor α) gene in the healthy women with a set of reproductive and nonreproductive BC risk factors. The breast tissues were collected from 120 cancer-free women who had undergone cosmetic mammoplasty. The genomic DNA was extracted from the breast tissues and the methylation level of PGR-α promoter CpG island was determined by using MeDIP-qPCR assay. Using regression analysis, we found that increasing menarche age is inversely associated with the high methylation of PGR-α promoter ( β = −0.790, SE = 0.362; P = 0.031). Although lactating women had more methylation than nonlactating women (P = 0.026, the t test), this result was not confirmed by regression models. Such an observation may be helpful in better understanding of the underlying mechanisms by which early age at menarche increases the risk of BC. However, this perspective requires further validations in larger studies of more subjects as well as the inclusion of other related genes.     

Visualization of Reinke’s crystals in normal and cryptorchid testis

Within the human testis, Reinke’s crystals are found in Leydig cells but their nature and function are poorly understood. The aim of our study was to investigate the properties of Reinke’s crystals in man with the normal morphology of the testis (control group) and infertile patients diagnosed with cryptorchidism. 20 biopsies from infertile patients and six biopsies from men with regular spermatogenesis (20–30 years.) were used. Sections of the testis tissue were stained with haematoxylin and eosin and a modified Masson’s method. Specimens were observed by bright field, confocal and transmission electron microscopy (TEM). The number of Reinke’s crystals in investigated groups was determined applying stereological methods. In both groups, Reinke’s crystals were noted within the cytoplasm and nuclei of Leydig cells. Some “free” crystals were found within the interstitial space, outside Leydig cells. Confocal microscopy proved to be very useful in the assessment of the shape and 3D reconstruction of the crystal. TEM analysis confirmed a hexagonal form of the crystal, while crystallographic data on sections of 70–300 nm thickness provided a better insight into the organization of the crystal lattice. Stereological analysis revealed a significant increase in the number of crystals in cryptorchid testes when compared with controls. Increased number of crystals in cryptorchid specimens leads to the assumption that the prolonged exposure to higher (abdominal) temperature might stimulate enzymes involved in the synthesis of the proteins of the crystal. However, the exact molecular nature of the crystal lattice remains in both normal and cryptorchid testis obscure.     

Localisation of esteroproteases in ‘resting’ salivary glands from different species and the effects of the organophosphorus inhibitor E600

Summary The distribution of reaction sites for esteroproteases has been assessed in resting salivary glands from rats, guinea-pigs, cats, dogs and rabbits using the new substrate N-acetyl-l-methionine -naphthyl ester (Lexow et al. 1979). Two main types of activity were observed in the parenchyma. 1) Condensed staining was present on the luminal sides of certain ducts in some, but not all, glands, and this type of staining was always most conspicuous in the submandibular glands of each species. It is likely that this periluminal esteroprotease activity arises from secretory enzymes that will pass into the saliva. Their greater presence in submandibular glands may have association with specialised licking activities. 2) Variable diffuse cytoplasmic staining was present in certain acinar cells from some, but not all, glands. This was most pronounced in the mucous cells of zygomatic glands from dogs. It is considered that the enzymes demonstrated in acinar cells are involved in processing secretory products rather than in being secretory themselves. The possibility that this includes signal peptidase is discussed. Ductal activity in rats, cats and guinea-pigs had some relationship with sites of tryptophan staining but not in rabbits or dogs. Any relationship with acinar staining was less evident. Mast cells, showing strong esteroprotease staining, were present in variable numbers in the different glands. Preliminary attempts to qualify the enzymes being demonstrated were made by using the inhibitor E600. This substance inhibited all activity in acinar cells from each species, and in rabbits and dogs it also inhibited the ductal activity as well. However, in rats and guinea-pigs it had little or no effect on the ductal activity and in cats it had only a small inhibitory effect on the ductal activity. E600 had no obvious inhibitory effects on mast cell activity.We are indebted to Professor O. von Deimling for the substrate, N-acetyl-l-methionine -naphthyl ester, used in this investigation     

Kinetics of degradation of ‘short-’ and ‘long-lived’ proteins in cultured mammalian cells

Two distinct classes of protein referred to as short- and long-lived (Poole and Wibo, 1973) were found in pulse-chased HeLa S-3 and BHK 21/C13 cells. From experiments with pulse times ranging from 1 min to 20 h, a clear inverse correlation emerged between the pulse length and the percentage of protein which was hydrolysed intracellularly in the first h of chase. Using a 5 min pulse labelling with ³H-leucine, cells were either harvested immediately or after a 2 h chase. Approximately 35% of the label incorporated by cells was lost in a 2h chase; however, electrophoretic separation of cytosol and residual cytoplasmic fractions revealed no significant alteration in their protein profiles. The technique of selectively labelling ‘short’ and ‘long-lived’ proteins, which implies qualitative differences between them, is more readily interpreted as an artificial polarisation of a declining statistical probability curve of proteolysis with time which is similar for all nascent proteins.