Models for intestinal fermentation: association between food components, delivery systems, bioavailability and functional interactions in the gut

There is increasing interest in the human colonic microbiota and in the way its metabolic activities impact on host health and well-being. For most practical purposes, however, the large bowel is inaccessible for routine investigation, and a variety of animal and in vitro model systems have been developed to study the microbiota. In vitro models range from simple closed systems using pure or defined mixed populations of bacteria, or faecal material, to more sophisticated complex multistage continuous cultures that are able to simulate many of the spatial, temporal and environmental attributes that characterize microbiological events in different regions of the large gut. Recent developments using these systems have enabled modelling of surface colonisation and biofilm development, a hitherto neglected area of study.     

Integrated communication between the nervous, endocrine and immune systems

Several data accumulated over recent years on the mechanisms underlying the interactions between the brain, hormones and the immune system. These data concern two major avenues of research: the evidence that brain-controlled, behavioral parameters can modulate the response of immunocompetent cells, and an increasing awareness that a number of chemical signals - neurotransmitters, hormones or mediators of immunity - are not, as previously believed, specific of given sets of tissues or of functions, but that, on the contrary, they can be produced and recognized by cellular elements belonging to any of those three systems. There is indeed evidence to indicate that signaling molecules involved in cellular communication are 'banalized': that means that their receptors are liable to be expressed in almost any tissue by a wide variety of cells. This statement, together with the discovery that intercellular regulation is multifactorial - that is, depends at any given time upon messages built up by combinations of signal molecules rather than by isolated transmitters - raises a certain number of theoretical problems as to the manner by which cells extract messages out of an important background noise. In the present paper, some of those theoretical problems will be presented in a summarized form, and their relevance for the interpretation of neuroendocrine or neuroimmunological interactions will be discussed.     

CLIP: a method for identifying protein-RNA interaction sites in living cells

Nucleic-acid binding proteins constitute nearly one-fourth of all functionally annotated human genes. Genome-wide analysis of protein-nucleic acid contacts has not yet been performed for most of these proteins, restricting attempts to establish a comprehensive understanding of protein function. UV cross-linking is a method typically used to determine the position of direct interactions between proteins and nucleic acids. We have developed the cross-linking and immunoprecipitation assay, which exploits the covalent protein-nucleic acid cross-linking to stringently purify a specific protein-RNA complex using immunoprecipitation followed by SDS-PAGE separation. In this way, the vast majority of non-specific contaminating RNA, which can bind to co-immunoprecipitated proteins or beads, can be removed. Here, we present an improved protocol that performs RNA linker ligation before the SDS-PAGE step, and describe its application to the specific purification and amplification of RNA ligands of Nova in neurons.     

The absence of a functional relationship between ATM and BLM, the components of BASC, in DT40 cells

Bloom syndrome (BS) and ataxia-telangiectasia (A-T) are rare autosomal recessive diseases associated with chromosomal instability. The genes responsible for BS and A-T have been identified as BLM and ATM, respectively, whose products were recently found to be components of BRCA1-associated genome surveillance complex (BASC), a supercomplex possibly involved in the recognition and repair of aberrant DNA structures. Based on experiments using BLM(-/-) DT40 cells and BLM(-/-)/RAD54(-/-) DT40 cells, we previously suggested that BLM functions to reduce the formation of double-strand breaks (DSBs) during DNA replication. To examine whether ATM is involved in the recognition and/or repair of DSBs generated in BLM(-/-) DT40 cells and to address the functional relationship between the two BASC components, we generated BLM(-/-)/ATM(-/-) DT40 cells and characterized their properties as well as those of ATM(-/-) and BLM(-/-) DT40 cells. BLM(-/-)/ATM(-/-) cells proliferated slightly more slowly than either BLM(-/-) or ATM(-/-) cells. The sensitivity of BLM(-/-)/ATM(-/-) cells to gamma-irradiation was similar to that of ATM(-/-) cells, while BLM(-/-) cells were slightly resistant to gamma-irradiation compared with wild-type cells. BLM(-/-) cells showed sensitivity to methyl methanesulfonate (MMS) and UV irradiation while ATM(-/-) cells did not show sensitivity to either agent. The sensitivity of BLM(-/-)/ATM(-/-) cells to MMS and UV was similar to that of BLM(-/-) cells. Disrupting the function of ATM reduced the targeted integration frequency in BLM(-/-) DT40 cells. However, a defect in ATM only slightly reduced the increased sister chromatid exchanges (SCEs) in BLM(-/-) DT40 cells.     

Functional richness, functional evenness and functional divergence: the primary components of functional diversity

Functional diversity is hypothesised as being beneficial for ecosystem functions, such as productivity and resistance to invasion. However, a precise definition of functional diversity, and hence a framework for its quantification, have proved elusive. We present a definition based on the analogy of the components of species diversity – richness, evenness and divergence. These concepts are applied to functional characters to give three components of functional diversity – functional richness, functional evenness and functional divergence. We demonstrate how each of these components may be calculated. It is hoped that our definition of functional diversity and its components will aid in elucidation of the mechanisms behind diversity/ecosystem-function relationships.     

Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells

In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells.     

Complexity of chemical communication in mammals: urinary components mediating sex discrimination by male guinea pigs

Male guinea pigs are more attracted to urine from female guinea pigs than to urine from males. Separation of urine into fractions characterized by molecular weight and functionality through several different methods established that this discrimination and preference is based on a pattern of components which have widely different chemical character. The active compounds range from macromolecules (MW >10,000) to small, relatively volatile molecules. These studies establish a complexity in chemical signals that has not been previously documented for any other species.     

Real-time observations of microtubule dynamic instability in living cells

Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations.     

Video microscopic observations of living,isolated embryo sacs of Nicotiana and their component cells

Summary Living embryo sacs and megagametophytic cells of Nicotiana alata and Nicotiana tabacum were obtained using enzymatic maceration and microdissection. The yields of isolated embryo sacs, egg apparatus and central cells were up to 35%, 40% and 35%, respectively. Vectorial movement of organelles and undulations of tubular structures, presumably endoplasmic reticulum, were observed in eggs, synergids and central cells using video-enhanced microscopy. Despite evident viability using the fluorochromatic reaction, the egg displays much less organelle movement and therefore appears to be quiescent. The large vacuole of the central cell is traversed by mobile strands of cytoplasm through which organelles migrate. A polygonal network is located at the periphery of the central cell, which may contribute to anchorage of the cell with the embryo-sac wall. The observation of organelle movement provides direct evidence of the condition of the cell and may be a useful approach for assessing cell vigor.     

Reassembly of living cells from dissociated components in Bryopsis

Protoplasm from Bryopsis maxima, a coenocytic green alga, wasdissociated into two fractions: chloroplasts, and protoplasmicfraction without chloroplasts (PF). The protoplasmic fraction(PF) included nuclei, mitochondria, dictyosomes, endoplasmicreticuli, etc. These two fractions were reassembled and formedprotoplasts, which developed into mature plants. (Received June 9, 1977; )     

Physical anthropology,peace and justice: Some observations

Human biological well being is a concern of physical anthropology. Genetic determinants and also sociocultural factors, which operate through biological bases, affect human biological quality. Several populations from different parts of the globe have been identified where varieties of detrimental genes occur in considerable numbers influencing health and some other related biological aspects of human life. Injustice will be done to those populations if necessary measures are not undertaken to improve their biological qualities. Peace cannot prevail in an atmosphere where people are deprived of the basic amenities for survival and maintenance of good health.     

Phase contrast observations on the endoplasmic reticulum of living cells in culture

Summary Phase contrast observation on a variety of cell types in culture revealed an extensive phase dark cytoplasmic network consisting of interconnected broad and fine branching trabeculae which extended from the perinuclear region to the cell margin; in structure, size and intracellular distribution, this network closely resembled the endoplasmic reticulum as seen in low power electron micrographs of unsectioned fixed cells of similar type. The networks of living cells were mobile and extraordinarily plastic. Both broad and fine network trabeculae underwent pronounced changes in shape, the broader elements sometimes extending into and partially merging with adjacent fine ones. The fine branching trabeculae altered in length and their junctions With other trabeculae continually shifted about; consequently individual trabeculae moved through the cytoplasmic matrix and the network pattern was forever changing. In some injured cells the networks appeared as highly mobile phase-light canaliculi which periodically opened into pathological vacuoles. The concept of the ER as a membranebo- und vacuolar system serving as an intracellular transport system was discussed in the light of the present findings.Aided in part by Grant GB-15 from the National Science Foundation administered by Dr. Donald E. Rounds.Eleanor Roosevelt Cancer Foundation Fellow — International Union Against Cancer (UICC).     

Coexisting peptides in hypothalamic neuroendocrine systems: Some functional implications

1. Coexisting with oxytocin or vasopressin in the cell bodies and nerve terminals of the hypothalamic-neurohypophysial system are smaller amounts of other peptides. For a number of these "copeptides" there is strong evidence of corelease with the major magnocellular hormones. Guided by the location of their specific receptors we have studied the effects of three copeptides, dynorphin, cholecystokinin (CCK), and corticotropin releasing hormone (CRH), on the secretion of oxytocin and vasopressin from isolated rat neural lobe or neurointermediate lobe preparations in vitro. 2. Dynorphin is coreleased with vasopressin from neural lobe nerve terminals and acts on neural lobe kappa-opiate receptors to inhibit the electrically stimulated secretion of oxytocin. Naloxone augments oxytocin release from the neural lobe in a manner directly proportional to the amount of vasopressin (and presumably dynorphin) released. 3. Cholecystokinin, coreleased with oxytocin by neural lobe terminals, has been shown to have high-affinity receptors located in the NL and to stimulate secretion of both oxytocin and vasopressin. CCK's secretagogue effect was independent of electrical stimulation and extracellular Ca2+ and was blocked by an inhibitor of protein kinase C. 4. CRH, coreleased with OT from the neural lobe, has receptors in the intermediate lobe of the pituitary, but not in the neural lobe itself. CRH stimulates the secretion of oxytocin and vasopressin from combined neurointermediate lobes but not from isolated neural lobes. Intermediate lobe peptides, alpha and gamma melanocyte stimulating hormone, induced secretion of oxytocin and vasopressin from isolated neural lobes. Their effect was, like that of CCK, independent of electrical stimulation and extracellular Ca2+ and blocked by an inhibitor of protein kinase C. 5. Among the CRH-producing parvocellular neurons of the paraventricular nucleus, in the normal rat, approximately half also produce and store vasopressin. After removal of glucocorticoid influence by adrenalectomy, virtually all of the CRH neurons contain vasopressin. 6. The two subtypes of CRH neurosecretory cells found in the normal rat possess different topographical distributions in the paraventricular nucleus, suggesting the possibility of differential innervation. Stress selectively activates the vasopressin containing subpopulation of CRH neurons, indicating that there are separate channels of regulatory input controlling the two components of the parvocellular CRH neurosecretory system.