Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

HLA-binding regions of HIV-1 proteins. I. Detection of seven HLA binding regions in the HIV-1 Nef protein.

The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4-18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose.     

Segment-specific changes with age in the expression of junctional proteins and the permeability of the blood-epididymis barrier in rats.

In aging Brown Norway rats, there is a striking increase in the number of halo cells in the epididymis; this reflects an activation of the immune system. As the blood-epididymis barrier should protect from immunological attack, we hypothesized that there would be changes in the structure and function of this barrier with age. To test this hypothesis, we assessed the immunocytochemical localization of occludin, ZO-1, and E-cadherin, as well as the lanthanum nitrate permeability of the blood-epididymis barrier, in the epididymides of Brown Norway rats aged 3, 18, and 24 mo. In the initial segment, occludin, ZO-1, and E-cadherin immunostaining was observed at the apico-lateral junction between principal cells in the 3-mo-old animals; with increasing age, occludin and ZO-1 reactivity decreased, while E-cadherin staining increased along the lateral membrane between principal cells. In the caput, corpus, and cauda epididymidis, occludin, ZO-1, and E-cadherin immunostaining showed segment-specific and age-dependent differences in their staining patterns. The most dramatic changes were seen in the corpus epididymidis with age; the intense E-cadherin cytoplasmic staining that was observed at 3 mo was absent by 24 mo, and no occludin or ZO-1 reactivity was observed in older animals. The greatest penetration of lanthanum nitrate across the blood-epididymis barrier and in the lumen was seen in the aging corpus epididymidis, while there was no barrier permeability in the initial segment or cauda epididymidis of the aged animals. Taken together, these data indicate that there are segment-specific decreases in the structural and functional integrity of the blood-epididymis barrier with age, most notably in the corpus epididymidis.     

Population variation of leaflet sleep movements in Oxalis grandis (Oxalidaceae)

To identify intraspecific variation in the expression of circadian leaflet movements, we observed changes in leaflet elevation angle in a controlled environment and in the field. Two morphologically and ecologically distinct populations of Oxalis grandis were compared: a typical mesic forest ecotype and an atypical, densely hirsute ecotype found on partially exposed shale outcroppings. Significant genotypic variation was detected within both populations. The controlled environment experiment revealed significant differences in the intrinsic rhythm between the two populations, primarily at the beginning and end of the photophase. We observed leaflet ascent and descent prior to lights-on and lights-off, respectively, processes we define as anticipatory. Hirsute ecotype plants showed a greater anticipation of lights-on, whereas the mesic ecotype plants showed a greater anticipation of lights-off. In the field, significant differences in leaflet elevation between the populations occurred in early morning and late afternoon, but not immediately preceding dusk. Contrary to the controlled environment experiment, mesic ecotype plants showed greater anticipation of dawn; plants from both populations showed similar anticipation of dusk. Plants of the hirsute ecotype exhibited higher sensitivity to sudden fluctuations in light level. We hypothesize that differences in light sensitivity partially explain the differences in the leaflet angle patterns between the field and controlled environments.