Instability of the cellular lipidome with age

The human lens nucleus is formed in utero, and from birth onwards, there appears to be no significant turnover of intracellular proteins or membrane components. Since, in adults, this region also lacks active enzymes, it offers the opportunity to examine the intrinsic stability of macromolecules under physiological conditions. Fifty seven human lenses, ranging in age from 12 to 82 years, were dissected into nucleus and cortex, and the nuclear lipids analyzed by electrospray ionization tandem mass spectrometry. In the first four decades of life, glycerophospholipids (with the exception of lysophosphatidylethanolamines) declined rapidly, such that by age 40, their content became negligible. In contrast the level of ceramides and dihydroceramides, which were undetectable prior to age 30, increased approximately 100-fold. The concentration of sphingomyelins and dihydrosphingomyelins remained unchanged over the whole life span. As a consequence of this marked alteration in composition, the properties of fiber cell membranes in the centre of young lenses are likely to be very different from those in older lenses. Interestingly, the identification of age 40 years as a time of transition in the lipid composition of the nucleus coincides with previously reported macroscopic changes in lens properties (e.g., a massive age-related increase in lens stiffness) and related pathologies such as presbyopia. The underlying reasons for the dramatic change in the lipid profile of the human lens with age are not known, but are most likely linked to the stability of some membrane lipids in a physiological environment.

Sphingolipid distribution changes with age in the human lens

The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens sections were initially analyzed by MALDI mass spectrometry imaging. A distinct annular distribution of the dihydrosphingomyelin, DHSM (d18:0/16:0), in the barrier region was observed in 64- and 70-year-old lenses but not in a 23-year-old lens. An increase in the dihydroceramide, DHCer (d18:0/16:0), in the lens nucleus was also observed in the older lenses. These findings were supported by ESI mass spectrometry analysis of lipid extracts from lenses dissected into outer, barrier, and nuclear regions. A subsequent analysis of 18 lenses ages 20-72 years revealed that sphingomyelin levels increased with age in the barrier region until reaching a plateau at approximately 40 years of age. Such changes in lipid composition will have a significant impact on the physical properties of the fiber cell membranes and may be associated with the formation of a barrier.-Deeley, J. M., J. A. Hankin, M. G. Friedrich, R. C. Murphy, R. J. W. Truscott, T. W. Mitchell, and S. J. Blanksby. Sphingolipid distribution changes with age in the human lens. J. Lipid Res. 2010. 51: 2753-2760.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Transcription factories : gene expression in unions?

Transcription is a fundamental step in gene expression, yet it remains poorly understood at a cellular level. Visualization of transcription sites and active genes has led to the suggestion that transcription occurs at discrete sites in the nucleus, termed transcription factories, where multiple active RNA polymerases are concentrated and anchored to a nuclear substructure. However, this concept is not universally accepted. This Review discusses the experimental evidence in support of the transcription factory model and the evidence that argues against such a spatially structured view of transcription. The transcription factory model has implications for the regulation of transcription initiation and elongation, for the organization of genes in the genome, for the co-regulation of genes and for genome instability.

KRAB zinc-finger proteins localise to novel KAP1-containing foci that are adjacent to PML nuclear bodies

The KRAB-zinc finger proteins (KRAB-ZFPs) represent a very large, but poorly understood, family of transcriptional regulators in mammals. They are thought to repress transcription via their interaction with KRAB-associated protein 1 (KAP1), which then assembles a complex of chromatin modifiers to lay down histone marks that are associated with inactive chromatin. Studies of KRAB-ZFP/KAP1-mediated gene silencing, using reporter constructs and ectopically expressed proteins, have shown colocalisation of both KAP1 and repressed reporter target genes to domains of constitutive heterochromatin in the nucleus. However, we show here that although KAP1 does indeed become recruited to pericentric heterochromatin during differentiation of mouse embryonic stem (ES) cells, endogenous KRAB-ZFPs do not. Rather, KRAB-ZFPs and KAP1 relocalise to novel nucleoplasmic foci that we have termed KRAB- and KAP1-associated (KAKA) foci. HP1s can also concentrate in these foci and there is a close spatial relationship between KAKA nuclear foci and PML nuclear bodies. Finally, we reveal differential requirements for the recruitment of KAP1 to pericentric heterochromatin and KAKA foci, and suggest that KAKA foci may contain sumoylated KAP1 - the form of the protein that is active in transcriptional repression.

Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μM lead chloride and 0.05 μM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 μM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 μM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μM and reached half-maximal inhibition of motility at about 50 μM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

The anatomy of the bill tip of kiwi and associated somatosensory regions of the brain : comparisons with shorebirds

Three families of probe-foraging birds, Scolopacidae (sandpipers and snipes), Apterygidae (kiwi), and Threskiornithidae (ibises, including spoonbills) have independently evolved long, narrow bills containing clusters of vibration-sensitive mechanoreceptors (Herbst corpuscles) within pits in the bill-tip. These ‘bill-tip organs’ allow birds to detect buried or submerged prey via substrate-borne vibrations and/or interstitial pressure gradients. Shorebirds, kiwi and ibises are only distantly related, with the phylogenetic divide between kiwi and the other two taxa being particularly deep. We compared the bill-tip structure and associated somatosensory regions in the brains of kiwi and shorebirds to understand the degree of convergence of these systems between the two taxa. For comparison, we also included data from other taxa including waterfowl (Anatidae) and parrots (Psittaculidae and Cacatuidae), non-apterygid ratites, and other probe-foraging and non probe-foraging birds including non-scolopacid shorebirds (Charadriidae, Haematopodidae, Recurvirostridae and Sternidae). We show that the bill-tip organ structure was broadly similar between the Apterygidae and Scolopacidae, however some inter-specific variation was found in the number, shape and orientation of sensory pits between the two groups. Kiwi, scolopacid shorebirds, waterfowl and parrots all shared hypertrophy or near-hypertrophy of the principal sensory trigeminal nucleus. Hypertrophy of the nucleus basorostralis, however, occurred only in waterfowl, kiwi, three of the scolopacid species examined and a species of oystercatcher (Charadriiformes: Haematopodidae). Hypertrophy of the principal sensory trigeminal nucleus in kiwi, Scolopacidae, and other tactile specialists appears to have co-evolved alongside bill-tip specializations, whereas hypertrophy of nucleus basorostralis may be influenced to a greater extent by other sensory inputs. We suggest that similarities between kiwi and scolopacid bill-tip organs and associated somatosensory brain regions are likely a result of similar ecological selective pressures, with inter-specific variations reflecting finer-scale niche differentiation.

Evidence for an Auditory Fovea in the New Zealand Kiwi (Apteryx mantelli).

Kiwi are rare and strictly protected birds of iconic status in New Zealand. Yet, perhaps due to their unusual, nocturnal lifestyle, surprisingly little is known about their behaviour or physiology. In the present study, we exploited known correlations between morphology and physiology in the avian inner ear and brainstem to predict the frequency range of best hearing in the North Island brown kiwi. The mechanosensitive hair bundles of the sensory hair cells in the basilar papilla showed the typical change from tall bundles with few stereovilli to short bundles with many stereovilli along the apical-to-basal tonotopic axis. In contrast to most birds, however, the change was considerably less in the basal half of the epithelium. Dendritic lengths in the brainstem nucleus laminaris also showed the typical change along the tonotopic axis. However, as in the basilar papilla, the change was much less pronounced in the presumed high-frequency regions. Together, these morphological data suggest a fovea-like overrepresentation of a narrow high-frequency band in kiwi. Based on known correlations of hair-cell microanatomy and physiological responses in other birds, a specific prediction for the frequency representation along the basilar papilla of the kiwi was derived. The predicted overrepresentation of approximately 4-6 kHz matches potentially salient frequency bands of kiwi vocalisations and may thus be an adaptation to a nocturnal lifestyle in which auditory communication plays a dominant role.

Neurobiological correlates of encoding strength of Pavlovian fear conditioned memory

Emotionally significant memories, especially those induced in conjunction with physical and mental trauma, are frequently retained for an individual’s lifetime. How these memories are organized and encoded within neural networks is a fundamental question. The lateral amygdala (LA) is a key nucleus for acquisition and maintenance of associative emotional memories. We used Pavlovian fear conditioning to study how ‘weaker’ and ‘stronger’ memories are encoded in neural networks of the LA. In Pavlovian fear conditioning a neutral stimulus, in this case a tone, is temporally paired with an aversive unconditioned stimulus (US), such as a foot shock. The previously neutral stimulus becomes a conditioned stimulus (CS) capable of eliciting defensive responses. We used time spent freezing when the CS is presented in a neutral context as a dependent variable measure of memory ‘strength’.

A recurrent network in the lateral amygdala: A mechanism for temporal coincidence detection

The dorsal lateral amygdala (LAd) is a vital nucleus for the formation of associations between aversive unconditioned stimuli (US) and neutral stimuli, such as auditory tones, which can become conditioned (CS) to the US through temporal pairing. Important aspects of CS-US associations are believed to occur within the LAd, however relatively little is known about the temporal behavior of local LAd networks. Information about the CS and US enters the LA via a rapid and direct thalamic input and a longer latency cortical path...

GABAA and NMDA receptors contribute to synaptic integration in lateral amygdala neurons

In classical fear conditioning a neutral conditioned stimulus (CS), is paired with an aversive unconditioned stimulus (US). The CS thereby acquires the capacity to elicit a fear response. This type of associative learning is thought to require co-activation of principal neurons in the lateral nucleus of the amygdala (LA) by two sets of synaptic inputs...

Amygdala-dependent learning increases the number of spinophilin-immunoreactive dendritic spines in the lateral amygdala

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons in the lateral nucleus of the amygdala (LA). One proposed form of cellular plasticity involves structural changes in the number and morphology of dendritic spines...

Differences in intrinsic properties of dorsally and ventrally located lateral amygdala neurons of the rat

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons of the lateral nucleus of the amygdala (LA). While the LA has been implicated as a key brain structure for fear learning, how its network of cellular components performs these operations is not yet known...

GABAa receptors contribute to synaptic integration in lateral amygdala neurons

In classical fear conditioning a neutral conditioned stimulus (CS) such as a tone, is paired with an aversive unconditioned stimulus (US) such as a shock. The CS thereby acquires the capacity to elicit a fear response. This type of associative learning is thought to require co-activation of principle neurons in the lateral nucleus of the amygdala (LA) by two sets of synaptic inputs, a weak CS and a strong US...