Kidney glomerular explants in serum-free media. Sequential morphologic and quantitative analysis of cell outgrowths

Guinea pig glomeruli were grown in vitro for 22 days in a serum-free medium composed of Waymouth's MB 752/1 supplemented with sodium pyruvate, nonessential amino acids, antibiotics, insulin, transferrin, selenium, triiodothyronine, and fibronectin (FN), and sequential morphologic and quantitative studies of cell outgrowth were performed. Glomeruli grown in serum-free medium showed preservation of glomerular visceral epithelial cells but extensive necrosis of endocapillary cells (endothelial and mesangial cells). Morphologic analysis demonstrated progressive morphologic changes in cultured glomerular cells; however, most cell types observed in culture appeared to grow from the epithelial side of the glomerular basement membrane. Mitosis was a prominent component of glomerular cell outgrowth in vitro, and total DNA increased slightly during glomerular culture. FN was required for glomerular cell outgrowth, and studies using FN fragments demonstrated that the carboxy-terminal portion of FN was required for whole glomerular attachment. These results are used to develop a model for glomerular cell outgrowth in vitro.     

Trypanosome hybrids generated in tsetse flies by nuclear fusion.

Genetic exchange may occur between two particular Trypanosoma brucei clones simultaneously transmitted by the same tsetse fly. We report here that this exchange takes place in the fly, through nuclear fusion. The resulting hybrids appear to be sub-tetraploid, some particular DNA sequences from one of the parental stocks being lost before enough cloned hybrid trypanosomes could be harvested for DNA analysis. A further reduction of the DNA content of these hybrids occurs gradually upon growth and yields near diploid value in a major part of the population. This mode of hybrid generation is different from the fusion of haploid gametes, which is thought to occur normally upon inoculation of metacyclic trypanosomes in their mammalian host. In this respect, the sub-tetraploid hybrids appear to undergo meiosis in the fly, generating sub-diploid metacyclic forms, then fusion in the mammalian blood.     

Body composition abnormalities in children with Prader-Willi syndrome and long-term effects of growth hormone therapy

Obesity and hypothalamic GH deficiency contribute in different ways to the disturbances of body composition in Prader-Willi syndrome (PWS); while both increase the fat compartment, the reduction of lean tissue mass has been attributed mainly to GH deficiency. Therefore, body composition measured by dual-energy X-ray absorptiometry was prospectively studied in 12 overweight children with PWS and weight for height (WfH) SDS >0 before and during 3.5 years of treatment with hGH (0.037 mg/kg/day) on average. In the long term, there is a net reduction of body fat from 3.1 to 1.2 SD, with a minimum at the end of the second year of treatment. WfH SDS correctly reflects body fat mass and its changes. The initial deficit of lean mass (-1.6 SD) is counteracted by GH only during the first year of therapy (increase to -1.25 SD). But in the long term, GH therapy does not further compensate for this deficit, when lean mass is corrected for its growth-related increase. In conclusion, exogenous GH changes the phenotype of children with PWS: fat mass becomes normal, but, at least in the setting studied, GH is not sufficient to normalize lean tissue mass.     

T cell responses against microsatellite instability-induced frameshift peptides and influence of regulatory T cells in colorectal cancer

High-level microsatellite-unstable (MSI-H) colorectal carcinomas (CRC) represent a distinct subtype of tumors commonly characterized by dense infiltration with cytotoxic T cells, most likely due to expression of MSI-H-related frameshift peptides (FSP). The contribution of FSP and classical antigens like MUC1 and CEA to the cellular immune response against MSI-H CRC had not been analyzed so far. We analyzed tumor-infiltrating and peripheral T cells from MSI-H (n = 4 and n = 14, respectively) and microsatellite-stable (MSS) tumor patients (n = 26 and n = 17) using interferon gamma ELISpot assays. Responses against 4 FSP antigens and peptides derived from MUC1 to CEA were compared with and without depletion of regulatory T cells, and the results were related to the presence of the respective antigens in tumor tissue. Preexisting FSP-specific T cell responses were detected in all (4 out of 4) tumor-infiltrating and in the majority (10 out of 14) of peripheral T cell samples from MSI-H CRC patients, but rarely observed in MSS CRC patients. Preexisting T cell responses in MSI-H CRC patients were significantly more frequently directed against FSP tested in the present study than against peptides derived from classical antigens MUC1 or CEA (p = 0.049). Depletion of regulatory T cells increased the frequency of effector T cell responses specific for MUC1/CEA-derived peptides and, to a lesser extent, T cell responses specific for FSP. Our data suggest that the analyzed FSP may represent an immunologically relevant pool of antigens capable of eliciting antitumoral effector T cell responses.     

Bacterial Uptake by the Marine Sponge <Emphasis Type="Italic">Aplysina aerophoba</Emphasis>

Sponges (Porifera) are filter feeders that take up microorganisms from seawater and digest them by phagocytosis. At the same time, many sponges are known to harbor massive consortia of symbiotic microorganisms, which are phylogenetically distinct from those in seawater, within the mesohyl matrix. In the present study, feeding experiments were performed to investigate whether phylogenetically different bacterial isolates, hereafter termed “food bacteria,” microbial seawater consortia, and sponge symbiont consortia are taken up and processed differently by the host sponge. Aplysina aerophoba retained high numbers of bacterial isolates and microbial seawater consortia with rates of up to 2.76 × 10⁶ bacteria (g sponge wet weight)^–1 h^–1, whereas the retention of sponge symbionts was lower by nearly two orders of magnitude [5.37 × 10⁴ bacteria (g sponge wet weight)⁻¹ h^–1]. In order to visualize the processing of a food bacterium within sponge tissues, the green fluorescent protein-labeled Vibrio strain MMW1, which had originally been isolated from A. aerophoba, was constructed. Incubation of this strain with A. aerophoba and subsequent visualization in tissue cryosections showed its presence in the choanocytes and/or endopinacocytes lining the canals but, unlike latex beads, not in deeper regions of the mesohyl, which suggests digestion of the bacteria upon contact with the host. Denaturing gradient gel electrophoresis (DGGE) was performed on the incubation seawater to monitor the changes in phylogenetic composition after incubation of the sponge with either seawater or sponge symbiont consortia. However, the DGGE experiment provided no evidence for selective processing of individual lineages by the host sponge. In conclusion, this study extends early studies by Wilkinson et al. (Proc R Soc London B 220:519–528, 1984) that sponges, here A. aerophoba, are able to differentiate between food bacteria and their own bacterial symbionts.     

Experience-dependent formation and recruitment of large vesicles from reserve pool

The sizes and contents of transmitter-filled vesicles have been shown to vary depending on experimental manipulations resulting in altered quantal sizes. However, whether such a presynaptic regulation of quantal size can be induced under physiological conditions as a potential alternative mechanism to alter the strength of synaptic transmission is unknown. Here we show that presynaptic vesicles of glutamatergic synapses of Drosophila neuromuscular junctions increase in size as a result of high natural crawling activities of larvae, leading to larger quantal sizes and enhanced evoked synaptic transmission. We further show that these larger vesicles are formed during a period of enhanced replenishment of the reserve pool of vesicles, from which they are recruited via a PKA- and actin-dependent mechanism. Our results demonstrate that natural behavior can induce the formation, recruitment, and release of larger vesicles in an experience-dependent manner and hence provide evidence for an additional mechanism of synaptic potentiation.     

A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains

We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent.     

DNA damage and biliary PAH metabolites in flatfish from Southern California bays and harbors, and the Channel Islands

Southern California bays and harbors have been shown to contain high concentrations of a variety of contaminants, including polycyclic aromatic hydrocarbons (PAHs), metals, and pesticides. Conventional monitoring tools do not assess exposure to PAHs in fish, or sublethal effects, which can be more sensitive indicators of stress than traditional methods. This study was conducted to evaluate DNA damage (a sublethal effect) and PAH exposure in flatfish from southern California bays and harbors, and the Channel Islands. California halibut (Paralichthys californicus) were collected from eight bays and harbors (Ventura Harbor, Channel Islands Harbor, Marina del Rey, King Harbor, Alamitos Bay, Long Beach Harbor, Newport Bay, San Diego Bay), and a reference site off Camp Pendleton. Pacific sanddab (Citharichthys sordidus) were collected near four of the Channel Islands (San Miguel Island, Santa Cruz Island, Anacapa Island, Santa Barbara Island). Metabolites of high molecular weight PAHs in fish bile were characterized using a semi-quantitative technique that measures fluorescent aromatic compounds (FACs) in fish bile. DNA damage in fish blood cells was assessed by measuring the amount of single-strand breaks in stained DNA using the Comet assay. The concentration of FACs in fish from all bays and harbors was elevated, with average concentrations ranging from three to ten times greater than FACs in reference fish. FAC levels were elevated in Pacific sanddabs from a Channel Islands station located between Santa Cruz Island and Anacapa Island. DNA damage varied by a factor of five among California halibut from bays and harbors, with significant damage occurring in fish from Alamitos Bay. There was a significant association between FAC concentrations and DNA damage in California halibut at Marina del Rey and Ventura Harbor, but not at other locations, presumably due to contaminants other than PAHs.