Biomedical subjects

Brian Herman

Publications and source records attributed to Brian Herman.

22 records · Page 2Linked to original sources

PubMed2002

Ageing and apoptosis.

Ageing is accompanied by a general decline of physiological function, especially at later stages, and significant increases in the incidence of cancer and other degenerative diseases. It has recently been hypothesized that alterations in apoptosis may contribute to these age-associated changes. However, whether there is a role for apoptosis in the ageing process and how ageing may modify the regulatory machinery of apoptosis remains obscure. Although the literature addressing these issues is scarce, research in this area is gaining momentum. Molecules involved in apoptosis signaling in mammals have been found to regulate ageing in organisms such as Caenorhabditis elegans and Drosophila melanogaster. Caloric restriction studies in a wide variety of organisms, ranging from yeast to mammals, suggest the conserved nature of the ageing regulatory systems. It seems very likely that signals that regulate ageing will impact apoptosis and the extent of apoptosis may then impact ageing. However, to date, there has been no direct evidence supporting the existence of such cross-communication between ageing and apoptosis in mammalian system. Here we review progress in the field.

Aging↗

PubMed2002

Age-associated increases in the activity of multiple caspases in Fisher 344 rat organs.

As organisms age, an increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling positive cells has been observed in a variety of tissues and cell types. However, whether this represents the increase of apoptosis has not been validated on molecular level. In this study we examined the endogenous activity of caspases that are known to be responsible for the execution of caspase-dependent apoptosis as a function of age in rat liver, lung, and spleen. We demonstrate that the extent of apoptosis in rat liver increases late during the aging process (i.e. 23-27 month) as indicated by the activation of executioner caspases-3, -6, and -7. We also found that the activity of caspase-3, -6, and -7 increased drastically in rat lung and spleen at late stages of aging. Despite reports that the level of Fas mRNA increases with age in rat liver and that Fas system regulates liver homeostasis, we did not detect activation of caspase-8, a key mediator of Fas-induced apoptosis, in aged liver. We also observed increased activities of two caspases, caspase-2 and caspase-9, which are involved in mitochondrion-mediated apoptosis in livers isolated from old rats, and found that hepatocytes isolated from old animals (>23 month) are more sensitive to oxidative stress that targets the mitochondria compared to those isolated from young (6 month) animals. Lastly, we demonstrate that the level of cytochrome c is lower in liver from old animals, probably as a result of expeditious degradation following its release into cytosol. Collectively, our results demonstrate that aging is associated with an increase in the activity of multiple caspases, suggesting that the extent of apoptosis increases as organs age. In the case of rat liver, this increase in caspase activation is more likely associated with the mitochondrial (i.e. intrinsic) pathway rather than the Fas-mediated caspase-8 (extrinsic) pathway of apoptosis.

Aging↗

PubMed2002

Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins.

To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.

Animals↗

PubMedDate not supplied

Multiphoton fluorescence lifetime contrast in deep tissue imaging: prospects in redox imaging and disease diagnosis.

Turbid tissues pose serious problems of strong absorption and scattering that make steady state fluorescence imaging methods less successful in imaging tissue layers deeper than a few tens of micrometers. Complications arise as one progresses from imaging cells to tissues to whole animal--which include enormous autofluorescence background in tissues and poor signal from regions of interest. Since the steady state, intensity-based methods cannot discriminate the photons arising from the fluorophores and the autofluorescence background, it is almost impractical to isolate these two signals. We describe multiphoton fluorescence lifetime imaging methods in the time domain to demonstrate fluorescence lifetime contrast in discriminating autofluorescence background from the fluorescent signals. Since the photophysical schemes of the fluorophore and autofluorescence contributions are distinct, it is feasible to isolate these two contributions in every pixel based only on their decay constants without compromising the SNR. We present preliminary lifetime measurements to characterize autofluorescence in various cell lines and ex vivo tissues obtained from mouse models. Together, these results suggest a novel direction in obtaining quantitative information from endogenous tissue fluorescence without any exogenous staining. The prospects for this approach in metabolic redox imaging and disease diagnosis are discussed.

Animals↗