Depth-Dependent Changes in Collagen Organization in the Human Peripapillary Sclera

PurposeThe collagen structure of the human peripapillary sclera plays a significant role in determining optic nerve head (ONH) biomechanics, and is therefore of interest in the study of glaucoma. The aim of the current work was to map the anisotropic collagen structure of the normal human peripapillary sclera as a function of tissue depth.MethodsWide-angle x-ray scattering was used to quantify collagen fibril orientation at 0.5mm intervals across six 150μm-thick serial sections through the peripapillary sclera of eight normal European-derived human eyes. Two structural parameters were measured: 1) the relative number of fibrils preferentially aligned at a given angle within the tissue plane, 2) the degree of collagen alignment (anisotropy).ResultsThe inner-most one-third of the peripapillary scleral stroma (nearest to the choroid) was characterised by collagen fibrils either randomly arranged or preferentially aligned radially with respect to the ONH. In contrast, the outer two-thirds of the tissue was dominated by a circumferential arrangement of collagen encircling the ONH. In all tissue regions the degree of collagen anisotropy peaked in the mid-stroma and progressively decreased towards the tissue surfaces, with the largest depth variations occurring in the inferior-nasal quadrant, and the smallest occurring in the superior-nasal quadrant.ConclusionsSignificant, region-specific variations in collagen structure are present in the human peripapillary sclera as a function of depth. In normal eyes, the circumferential collagen fibril architecture is most prominent in the outer two-thirds of the stroma, possibly as a mechanical adaption to more effectively support the lamina cribrosa at the level of its insertion into the scleral canal wall.     

Cyclooxygenase-2 transactivates the epidermal growth factor receptor through specific E-prostanoid receptors and tumor necrosis factor-alpha converting enzyme

Cyclooxygenase-2 is often highly expressed in epithelial malignancies and likely has an active role in tumor development. But how it promotes tumorigenesis is not clearly defined. Recent evidence suggests that this may involve transactivation of the epidermal growth factor receptor through E-prostanoid receptors, but reports differ about the mechanism by which this occurs. We found that E-prostanoid receptors 2-4, but not 1, transactivated the epidermal growth factor receptor. This required metalloproteinase activity, leading to release of growth factors from the cell surface. Both transforming growth factor-alpha and amphiregulin were released in response to over-expression of cyclooxygenase-2, but betacellulin and heparin-binding EGF-like growth factor were not. The metalloproteinase tumor necrosis factor-alpha converting enzyme was required for proteolytic release of transforming growth factor-alpha. We also found that addition of epidermal growth factor receptor ligands to HEK293 cells induced cyclooxygenase-2 expression, suggesting that by activating epidermal growth factor receptor signaling, cyclooxygenase-2 potentially creates a self-perpetuating cycle of cell growth. Consistent with this, inhibition of cyclooxygenase-2 reduced growth of epidermal growth factor receptor over-expressing MCF-10A breast epithelial cells in three-dimensional culture.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

Restoration of norepinephrine release,cognitive performance,and dendritic spines by amphetamine in aged rat brain

Age-related dysfunctions in specific neurotransmitter systems likely play an important role in cognitive decline even in its most subtle forms. Therefore, preservation or improvement of cognition via augmentation of neurotransmission is a potential therapeutic strategy to prevent further cognitive deficits. Here we identified a particular neuronal vulnerability in the aged Fischer 344 rat brain, an animal model of neurocognitive aging. Specifically, we demonstrated a marked impairment in glutamate-stimulated release of norepinephrine (NE) in the hippocampus and cerebral cortex of aged rats, and established that this release was mediated by N-methyl-D -aspartate (NMDA) receptors. Further, we also demonstrated that this decrease in NE release is fully rescued by the psychostimulant drug amphetamine (AMPH). Moreover, we showed that AMPH increases dendritic spine maturation, and importantly shows preclinical efficacy in restoring memory deficits in the aged rat through its actions to potentiate NE neurotransmission at β-adrenergic receptors. Taken together, our results suggest that deficits in glutamate-stimulated release of NE may contribute to and possibly be a determinant of neuronal vulnerability underlying cognitive decline during aging, and that these deficits can be corrected with currently available drugs. Overall these studies suggest that repurposing of psychostimulants for age-associated cognitive deficits is a potential avenue to delay or prevent cognitive decline and/or frank dementia later in life.     

Genetic Characterization of Plasmids Containing Genes Encoding Enzymes of Leucine Biosynthesis in Endosymbionts (Buchnera) of Aphids

The prokaryotic endosymbionts (Buchnera) of aphids are known to provision their hosts with amino acids that are limiting in the aphid diet. Buchnera from the aphids Schizaphis graminum and Diuraphis noxia have plasmids containing leuABCD, genes that encode enzymes of the leucine biosynthetic pathway, as well as genes encoding proteins probably involved in plasmid replication (repA1 and repA2) and an open reading frame (ORF1) of unknown function. The newly reported plasmids closely resemble a plasmid previously described in Buchnera of the aphid Rhopalosiphum padi [Bracho AM, Martínez-Torres D, Moya A, Latorre A (1995) J Mol Evol 41:67–73]. Nucleotide sequence comparisons indicate conserved regions which may correspond to an origin of replication and two promoters, as well as inverted repeats, one of which resembles a rho-independent terminator. Phylogenetic analyses based on amino acid sequences of leu gene products and ORF1 resulted in trees identical to those obtained from endosymbiont chromosomal genes and the plasmid-borne trpEG. These results are consistent with a single evolutionary origin of the leuABCD-containing plasmid in a common ancestor of Aphididae and the lack of plasmid exchange between endosymbionts of different aphid species. Trees for ORF1 and repA (based on both nucleotides and amino acids) are used to examine the basis for leu plasmid differences between Buchnera of Thelaxes suberi and Aphididae. The most plausible explanation is that a single transfer of the leu genes to an ancestral replicon was followed by rearrangements. The related replicon in Buchnera of Pemphigidae, which lacks leuABCD, appears to represent the ancestral condition, implying that the plasmid location of the leu genes arose after the Pemphigidae diverged from other aphid families. This conclusion parallels previously published observations for the unrelated trpEG plasmid, which is present in Aphididae and absent in Pemphigidae. Recruitment of amino acid biosynthetic genes to plasmids has been ongoing in Buchnera lineages after the infection of aphid hosts. Received: 9 March 1998 / Accepted: 18 May 1998.     

Prevalence of Escherichia coli in surface waters of Southeast Asian cities

Surface water samples were collected from rivers which fed into large urban areas within Vietnam, Indonesia, Cambodia, and Thailand and were processed to enumerate Escherichia coli. Selected isolates were further characterized using PCR to detect the presence of specific virulence genes. Analyzing the four countries together, the approximate mean cfu/100 ml for E. coli counts in the dry season were log 4.3, while counts in the wet season were log 2.8. Of the 564 E. coli isolates screened for the presence of pathogenic genes, 3.9 % possessed at least one virulence gene. The most common pathogenic types found were Shiga toxin-producing E. coli isolates. These results reinforce the importance of monitoring urban surface waters for fecal contamination, that E. coli in these water environments may serve as opportunistic pathogens, and may help in determining the impact water usage from these rivers have on the public health of urban populations in Southeast Asia.     

Traditional Aboriginal Preparation Alters the Chemical Profile of Carica papaya Leaves and Impacts on Cytotoxicity towards Human Squamous Cell Carcinoma

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation.     

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity.