Search

Lysosomes are a major organelle for degrading macromolecules. When deprived of nutrients, cells activate the autophagy and lysosome biogenesis pathways to recycle cytoplasmic materials and to increase lysosomal degradation capacity for survival, respectively. We have identified a condition in which cells accumulated enlarged lysosomes upon starvation and lysosome inhibition. Selective autophagy and inhibition of the mechanistic target of rapamycin (mTOR) in combination with lysosome inhibition were not able to induce this phenomenon. Conversely, knocking out autophagy genes, ATG5 or ATG7, had no effects on the enlarged lysosome formation. This suggests that the enlarged lysosome formation is an autophagy independent process. Remarkably, adding glutamine to the treatment can prevent formation of the enlarged lysosomes and dissipate the pre-existing ones. Furthermore, the nucleus/cytoplasm translocation of the transcription factor EB (TFEB), but not mTOR activity, correlates with the formation/dissipation of enlarged lysosomes. Knockdown of TFEB, however, suggests that TFEB-mediated lysosome biogenesis is not directly involved in the process. These results indicate that there is a novel mechanism by which lysosome homeostasis can be regulated under certain stress conditions.

This study aims to understand the effect of milk protein concentrate (MPC80) solubility on its susceptibility to initiate fouling on stainless steel (SS) surfaces during thermal processing. In order to generate powders with different dissolution characteristics, a part of powder was stored at 25 °C (well-soluble powder) and the remaining part was stored at 40 °C (poor-soluble powder) for 2 weeks. Fouling characteristics of reconstituted MPC80 powder with 80 g protein per 100 g powder were studied using a custom-built benchtop plate heat exchanger. Exposing the MPC80 powder to a higher temperature during storage (40 °C) significantly decreased the solubility and increased the amount of foulant on SS coupons (P

Unlike sporadic daytime heat spikes, a consistent increase in night-time temperatures can potentially derail the genetic gains being achieved. Ten winter wheat genotypes were exposed to six different night-time temperatures (15-27°C) during flowering and grain-filling stages in controlled environment chambers. We identified the night-time temperature of 23o C as the critical threshold beyond which a consistent decline in yields and quality was observed. Confocal laser scanning micrographs of central endosperm, bran, and germ tissue displayed differential accumulation of protein, lipid, and starch with increasing night-time temperatures. KS07077M-1 recorded a decrease in starch and an increase in protein and lipid in central endosperm with increasing night-time temperatures, whereas the same was significantly lower in the tolerant SY Monument. Expression analysis of genes encoding 21 enzymes (including isoforms) involved in grain-starch metabolism in developing grains revealed a high night-time temperature (HNT)-induced reduction in transcript levels of adenosine diphosphate glucose pyrophosphorylase small subunit involved in starch synthesis and a ≥2-fold increase in starch degrading enzymes isoamylase III, alpha-, and beta-amylase. The identified critical threshold, grain compositional changes, and the key enzymes in grain starch metabolism that lead to poor starch accumulation in grains establish the foundational knowledge for enhancing HNT tolerance in wheat.

In sorghum (Sorghum bicolor [L.] Moench), the impact of heat stress during flowering on seed set is known, but mechanisms that lead to tolerance are not known. A diverse set of sorghum genotypes was tested under controlled environment and field conditions to ascertain the impact of heat stress on time-of-day of flowering, pollen viability, and ovarian tissue. A highly conserved early morning flowering was observed, wherein >90% of spikelets completed flowering within 30 min after dawn, both in inbreds and hybrids. A strong quantitative impact of heat stress was recorded before pollination (reduced pollen viability) and post pollination (reduced pollen tube growth and linear decline in fertility). Although viable pollen tube did reach the micropylar region, 100% spikelet sterility was recorded under 40/22°C (day/night temperatures), even in the tolerant genotype Macia. Heat stress induced significant damage to the ovarian tissue near the micropylar region, leading to highly condensed cytoplasmic contents and disintegrated nucleolus and nucleus in the susceptible genotype RTx430. Whereas, relatively less damages to ovarian cell organelles were observed in the tolerant genotype Macia under heat stress. Integrating higher tolerance in female reproductive organ will help in effective utilization of the early morning flowering mechanism to enhance sorghum productivity under current and future hotter climate.

Ehrlichia chaffeensis, a tick-transmitted rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis. Biochemical characterization of this and other related Rickettsiales remains a major challenge, as they require a host cell for their replication. We investigated the use of an axenic medium for E. chaffeensis growth, assessed by protein and DNA synthesis, in the absence of a host cell. E. chaffeensis organisms harvested from in vitro cultures grown in a vertebrate cell line were fractionated into infectious dense-core cells (DC) and the non-infectious replicating form, known as reticulate cells (RC) by renografin density gradient centrifugation and incubated in the axenic medium containing amino acids, nucleotides, and different energy sources. Bacterial protein and DNA synthesis were observed in RCs in response to glucose-6-phosphate, although adenosine triphosphate, alpha-ketoglutarate or sodium acetate supported protein synthesis. The biosynthetic activity could not be detected in DCs in the axenic medium. While the data demonstrate de novo protein and DNA synthesis under axenic conditions for E. chaffeensis RCs, additional modifications are required in order to establish conditions that support bacterial replication, and transition to DCs.

Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated 'gcs-1') were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development.

ADDITIONAL INDEX WORDS. Cuphea llavea, Ipomoea batatas, oedema, intumescence, Pelargonium, Solanum lycopersicum ABSTRACT. Many plant species are prone to physiological disorders in which lesions develop on the leaf tissue. Nomenclature for such lesions has included intumescences, excrescences, neoplasms, galls, genetic tumors, enations, and oedemata. Interchangeably using these terms causes confusion as to whether these names refer to the same or different disorders. Two of the most commonly used names are oedema and intumescence. The objective of this research was to characterize the development of lesions on ornamental sweetpotato (Ipomoea batatas 'Blackie'), tomato (Solanum lycopersicum 'Maxifort'), interspecific hybrid geranium (Pelargonium · 'Caliente Coral'), and bat-faced cuphea (Cuphea llavea 'Tiny Mice') to determine similarities and differences in morphology and nomenclature among these physiological disorders. Light microscopy was used to characterize differences in cross-sectional height, width, and area of lesions on each species. Additionally, leaf tissue samples were embedded in paraffin, and 10-mm cross-sections were stained with Toluidine blue O and observed using light microscopy to identify specific cell layers involved with lesion development. Field emission scanning electron microscopy (SEM) and digital photography were used to observe the microscopic and macroscopic stages of lesion development, respectively, on each species. The lesions observed on ornamental sweetpotato were significantly greater in height and area than on the other three species, whereas tomato lesions were significantly greater in width. Lesions on ornamental sweetpotato and bat-faced cuphea occurred predominantly on the adaxial surface of the leaf, whereas lesions on geranium and tomato occurred predominantly on the abaxial surface. With lesions on tomato, ornamental sweetpotato, and bat-faced cuphea, the epidermis was often subjected to the same hypertrophy apparent in the underlying parenchyma cells, ultimately allowing for greater cell expansion. However, in geranium, the epidermis resisted the expansion of the underlying cells, resulting in the eventual tearing of this tissue layer. Previous research indicates that lesion development on geranium is closely related to water status within the plant and may result in a wound response or provide a means of facilitated gas exchange. On the contrary, development of lesions on ornamental sweetpotato and tomato is believed to involve light quality. Based on these results and observations, two disorders occur across these species. The term ''intumescence'' should be used when referring to abnormal lesions on ornamental sweetpotato and tomato, and the term ''oedema'' should be used when referring to lesions on geranium. The term ''intumescence'' should also be used when referring to bat-faced cuphea lesions resulting from the morphological and anatomical aspects of these lesions closely resembling development on ornamental sweetpotato and tomato. Future research should investigate the role of light quality regarding development on this species.

Control of salivary secretion in ticks involves autocrine dopamine activating two dopamine receptors: D1 and Invertebrate-specific D1-like dopamine receptors. In this study, we investigated Na/K-ATPase as an important component of the secretory process. Immunoreactivity for Na/K-ATPase revealed basal infolding of lamellate cells in type-I, abluminal interstitial (epithelial) cells in type-II and labyrinth-like infolding structures opening towards the lumen in type-III acini. Ouabain (10 μmol l−1), a specific inhibitor of Na/K-ATPase, abolished dopamine-induced salivary secretion by suppressing fluid transport in type III acini. At 1 μmol l−1, ouabain, the secreted saliva was hyperosmotic. This suggests that ouabain also inhibits an ion resorptive function of Na/K-ATPase in the type I acini. Dopamine/ouabain were not involved in activation of protein secretion, while dopamine-induced saliva contained constitutively basal level of protein. We hypothesize that the dopamine-dependent primary saliva formation, mediated by Na/K-ATPase in type III and type II acini, is followed by a dopamine-independent resorptive function of Na/K-ATPase in type I acini located in the proximal end of the salivary duct.

During each molting cycle of insect development, synthesis of new cuticle occurs concurrently with the partial degradation of the overlying old exoskeleton. Protection of the newly synthesized cuticle from molting fluid enzymes has long been attributed to the presence of an impermeable envelope layer that was thought to serve as a physical barrier, preventing molting fluid enzymes from accessing the new cuticle and thereby ensuring selective degradation of only the old one. In this study, using the red flour beetle, Tribolium castaneum , as a model insect species, we show that an entirely different and unexpected mechanism accounts for the selective action of chitinases and possibly other molting enzymes. The molting fluid enzyme chitinase, which degrades the matrix polysaccharide chitin, is not excluded from the newly synthesized cuticle as previously assumed. Instead, the new cuticle is protected from chitinase action by the T. castaneum Knickkopf (TcKnk) protein. TcKnk colocalizes with chitin in the new cuticle and organizes it into laminae. Down-regulation of TcKnk results in chitinase-dependent loss of chitin, severe molting defects, and lethality at all developmental stages. The conservation of Knickkopf across insect, crustacean, and nematode taxa suggests that its critical roles in the laminar ordering and protection of exoskeletal chitin may be common to all chitinous invertebrates.

Transmission electron microscopy (TEM) of hygroscopic, permeable, and electron-absorbing biological cells has been an important challenge due to the volumetric shrinkage, electrostatic charging, and structural degradation of cells under high vacuum and fixed electron beam.(1-3) Here we show that bacterial cells can be encased within a graphenic chamber to preserve their dimensional and topological characteristics under high vacuum (10(-5) Torr) and beam current (150 A/cm(2)). The strongly repelling π clouds in the interstitial sites of graphene's lattice(4) reduces the graphene-encased-cell's permeability(5) from 7.6-20 nm/s to 0 nm/s. The C-C bond flexibility(5,6) enables conformal encasement of cells. Additionally, graphene's high Young's modulus(6,7) retains cell's structural integrity under TEM conditions, while its high electrical(8) and thermal conductivity(9) significantly abates electrostatic charging. We envision that the graphenic encasement approach will facilitate real-time TEM imaging of fluidic samples and potentially biochemical activity.

Maize prolamin (zein), together with starch, hydroxypropyl methylcellulose, sugar, salt, yeast and water can form wheat-like cohesive, extensible, viscoelastic dough when mixed above room temperature (e.g. 40 � C). This dough is capable of holding gas. However, it is excessively extensible, and when used for hearth-type rolls, it tends to become flat. Bench-scale defatting of zein with chloroform at room temperature significantly (P

Gluten-free bread was prepared from commercial zein (20 g), maize starch (80 g), water (75 g), saccharose, NaCl and dry yeast by mixing above zein's glass transition temperature (Tg) at 40°C. Addition of hydroxypropyl methylcellulose (HPMC, 2 g) significantly improved quality, and the resulting bread resembled wheat bread having a regular, fine crumb grain, a round top and good aeration (specific volume 3.2 ml/g). In model studies, HPMC stabilized gas bubbles well. Additionally, laser scanning confocal microscopy (LSCM) revealed finer zein strands in the dough when HPMC was present, while dynamic oscillatory tests showed that HPMC rendered gluten-like hydrated zein above its Tg softer (i.e. |G*| was significantly lower). LSCM revealed that cooling below Tg alone did not destroy the zein strands; however, upon mechanical impact below Tg, they shattered into small pieces. When such dough was heated above Tg and then remixed, zein strands did not reform, and this dough lacked resistance in uniaxial extension tests. When within the breadmaking process, dough was cooled below Tg and subsequently reheated, breads had large void spaces under the crust. Likely, expanding gas bubbles broke zein strands below Tg resulting in structural weakness.

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE − mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE − mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases—GelE and SprE—is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.

C2D macrophage cells protect immunocompromised mice from experimentally induced pneumonias after intraperitoneal (i.p.) adoptive transfer. These macrophage cells are immature and display minimal activity in vitro. Therefore, we wanted to understand how adoptive transfer affected these cells. We believe that the in vivo environment affects the phenotypic and functional characteristics of macrophages that help maintain the physiological integrity of the host. To test this hypothesis, we characterized the trafficking patterns and cellular changes of the established macrophage C2D cell line after adoptive transfer. We examined phenotypic changes of the C2D macrophage cells in vivo with and without stimulation with gamma interferon (IFN-γ). After in vivo i.p. adoptive transfer, C2D macrophage cells trafficked to the lungs, spleen, lymph nodes, and bone marrow of recipient mice. The cells were detected for as long as 2 months, and the cells expressed increased levels of CD11b, c-fms, and F4/80 on their surface, becoming more differentiated macrophages compared to cells maintained in vitro. Upon in vivo stimulation with IFN-γ, c-fms levels decreased while Gr-1 levels increased compared to in vivo, unstimulated, phosphate-buffered saline-injected controls. These responses were independent of the genetic backgrounds of the recipient mice. These data support the hypothesis and indicate that C2D macrophage cells respond to in vivo signals that are absent during in vitro culture.

Nine grain sorghum cultivars with a broad range of ethanol fermentation efficiencies were selected to characterize the changes in sorghum protein in digestibility, solubility, and microstructure during mashing and to relate those changes to ethanol fermentation quality of sorghum. Mashing reduced in vitro protein digestibility considerably, and a large amount of polymers cross-linked by disulfide bonds were developed during mashing. As a marker of cross-linking, protein digestibility of the original samples was highly related to conversion efficiency. gamma-Kafirin (%) neither correlated to ethanol yield nor conversion efficiency significantly. Solubility of proteins in an alkaline borate buffer in conjunction with SDS decreased substantially after mashing. Solubility and the SE-HPLC area of proteins extracted from mashed samples were highly correlated with ethanol fermentation. Ethanol yield increased and conversion efficiency improved notably with the increase of extracted proteins from mashed samples. SE-HPLC total area could be used as an indicator to predict ethanol fermentation. CFLSM images proved that sorghum proteins tended to form highly extended, strong web-like microstructures during mashing. The degree of protein cross-linking differed among samples, and more open microstructures were observed in samples with higher conversion efficiencies. The web-like protein matrix was found to hold not only starch granules but also some oligosaccharides or polysaccharides inside. The formation of web-like microstructures because of cross-linking reduced conversion efficiency.

Sorghum ( Sorghum bicolor (L.) Moench ) is a starch-rich grain similar to maize ( Zea mays L.), but sorghum has been underutilized for biobased products and bioenergy. This study was designed to investigate the effects of supercritical-fluid-extrusion (SCFX) of sorghum on ethanol production. Morphology, chemical composition, and thermal properties of extruded sorghum were characterized. Analysis of extruded sorghum showed increased measurable starch content, free sugar content, and high levels of gelatinized starch. SCFX cooked and non-extruded sorghum were further liquefied, saccharified, and fermented to ethanol by using Saccharomyces cervisiae . The ethanol yield increased as sorghum concentration increased from 20 to 40% for both extruded and non-extruded sorghum. Ethanol yields from SCFX cooked sorghum were significantly greater than that from non-extruded sorghum (>5%).

Adiponectin (Acrp30) is an insulin-sensitizing hormone produced and secreted exclusively by adipose tissue. Confocal fluorescent microscopy demonstrated the colocalization of adiponectin with the Golgi membrane markers p115, beta-COP, and the trans-Golgi network marker, syntaxin 6. Treatment of cells with brefeldin A redistributed adiponectin to the endoplasmic reticulum where it colocalized with the chaperone protein BIP and inhibited secretion of adiponectin demonstrating a requirement for a functional Golgi apparatus for adiponectin release. Confocal fluorescent microscopy also demonstrated a colocalization of endogenous adiponectin with that of expressed GGA1myc (Golgi-localizing gamma-adaptin ear homology ARF-binding protein) but with no significant overlap between adiponectin and the GGA2myc or GGA3myc isoforms. Consistent with confocal fluorescent microscopy, transmission electron microscopy demonstrated the colocalization of GGA1 with adiponectin. Although GGA1 did not directly interact with the adiponectin protein, the adiponectin enriched membrane compartments of adipocyte were precipitated by a GST-GGA1 cargo binding domain (VHS) fusion protein but not with a GST-GGA2 VHS or GST-GGA3 VHS fusion proteins. Moreover, co-expression of adiponectin with a GGA1 dominant-interfering mutant (GGA1-VHS GAT domain) resulted in a marked inhibition of adiponectin secretion in both 3T3L1 adipocytes and HEK293 cells, whereas no inhibition was detected with the truncated mutants GGA2-VHSGAT or GGA3-VHSGAT. Moreover, co-expression of wild type GGA1 with adiponectin enhanced secretion of adiponectin. Interestingly, leptin secretion was unaffected by neither the wild type form or GGA1 mutant. Taken together these data demonstrate that the trafficking of adiponectin through its secretory pathway is dependent on GGA-coated vesicles.

Previous studies have demonstrated that asparagine-143 of gammaS crystallin is preferentially deamidated in human cataractous lenses. A possible mechanism of nonenzymatic deamidation involves the initial formation of L -succinimidyl and D -succinimidyl intermediates, followed by ring cleavage and production of the respective L - and D - forms of beta-aspartate and alpha-aspartate. To determine if this mechanism occurs during cataractogenesis of the human lens, synthetic peptide standards together with reverse phase HPLC were used to purify and quantitate tryptic peptides from cataractous lenses that contained the beta-aspartate and alpha-aspartate forms of residue 143. For all lenses analysed, the ratios of beta-aspartate/alpha-aspartate produced were consistent with a mechanism of deamidation involving succinimide intermediates. Surprisingly, the D / L ratios for all beta- and alpha-aspartates produced were very low, suggesting that the mechanism of deamidation preferentially involves the L -succinimidyl intermediate, with formation of the D -succinimidyl intermediate almost completely blocked by steric hindrance. Together, the results demonstrate that deamidation of asparagine-143 from gammaS crystallin of different human cataracts undergoes the same mechanism, which due to higher ordered structure of the native protein, results in the preferential production of one of two possible succinimidyl intermediates.

We have previously shown that biologically uncommon d-beta-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of alpha A-crystallin from aged human lenses. The amounts increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-beta-aspartic acids in alpha A-crystallin and cataract formation, we measured the d/l ratio of beta-Asp-151 of alpha A-crystallin from both cataractous and age-matched normal human lenses. alpha A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the alpha- and beta-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of beta-Asp-151 of alpha A-crystallin from normal lenses is not statistically significant from that of alpha A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.

It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as gamma-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from gamma-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation.

The purpose of the current study was to localize the lens membrane protein, MIP 26, in nuclear fiber cells from different regions of aged normal and age-related cataractous human lenses. Adult, juvenile, fetal and embryonic nuclear regions in aged normal and age-related nuclear cataractous lenses were morphologically and biochemically characterized using the technologies of immuno-gold (5 nm) labeling, semi-thin sections (200-500 nm), serial sections, DiI staining following by photobleaching, transmission electron microscopy and spot-blot analysis. Numbers of gold particles per micron length of plasma membrane and numbers of gold particles per square micron of cytosol in the embryonic-fetal and juvenile-adult nuclear regions were quantified. Results showed that the labeling pattern of MIP 26 localized to the cytosol was unique to senescent fiber cells from age-related cataractous lenses. Numbers of gold particles per square micron of cytosol in the embryonic-fetal nucleus of age-related cataractous lenses were significantly elevated (P0.001) above numbers from fiber cells located within the adult or juvenile nuclei of the same lens or senescent fiber cells from aged normal lenses. Some of the cytosolic labeling in cataracts was localized to lipid vesicles, while the remaining labeling was negative for the lipid specific stain DiI. Spot blot analysis demonstrated that binding of the ant-MIP 26 serum was exclusive to large molecular weight components greater than 10 kDa, and not to small molecular weight fragments of the protein. The results of the current study supply further evidence that damage to membranes occurs in senescent fiber cells during age-related nuclear cataracts, resulting in the internalization of structures containing the membrane protein MIP 26.

Although it has been hypothesized that age-dependent deamidation of glutamine and/or asparagine residues may play an important role in the turnover of proteins in vivo, surprisingly little is known concerning the extents of deamidation of biologically important proteins with very long half-lives. Alpha-A crystallin is the most abundant protein of the adult human lens, which contains long-lived proteins in the central fetal-embryonic region that were synthesized before birth of the individual. Peptides, corresponding to tryptic fragments of alpha-A crystallin, were synthesized with either the expected glutamine-6, glutamine-50, and glutamine-147 residues, or deamidated glutamic acid residues at the same positions. These synthetic peptides were used to identify and quantitate the amidated versus deamidated forms of each tryptic fragment of alpha-A crystallin from the fetal-embryonic region of lenses from donors of increasing age up to 64 years old. The results demonstrate that all three glutamine residues are very stable, with glutamine-50 undergoing a maximum of approximately 30% deamidation after 64 years postsynthesis, while glutamine-6 and glutamine-147 undergo no detectable deamidation during the same period of time. Together, these results are consistent with the hypothesis that resistance to age-dependent, nonenzymatic deamidation may be an important prerequisite for the stability of proteins in vivo.

To corroborate the findings of finger-like membrane projections in monkey and baboon lenses, in human lens fiber cells.Normal human lenses, two months to 76 years old, as well as age-related nuclear cataracts, were immersion fixed in 2% paraformaldehyde-0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 24 h at room temperature, cut into 200-500 microm thick sections along the equatorial axis, fixed for an additional 12-18 h at room temperature, dehydrated in an ascending ethanol series and embedded in Lowicryl K4M. Semi-thick sections (0.25-1.0 microm) were cut, absorbed onto 75 or 100 nickel slotted grids, labeled with anti-MIP 26 or phalloidin, stained with 2% uranyl acetate and viewed by transmission electron microscopy at 100 kV.Transmission electron microscopy micrographs demonstrated the presence of finger-like plasma membrane projections measuring 0.16-0.25 microm in diameter and 1.0-6.5 microm in length with bulbous terminal tips in the most senescent fiber cells in two-month and older normal lenses, as well as, in nuclear cataracts. These projections appeared to overlie furrowed membrane domains in the extracellular space, as well as project into the cytosol along the cytosolic leaflet of plasma membrane.The results extend the findings in monkey and baboon lenses, to the human lens, and demonstrate that these projections, which sparsely label with antiserum against MIP 26, but not filamentous actin, not only extend into the extracellular space, but also project inward into the cytosol.

Improved control of vector-borne diseases requires an understanding of the molecular factors that determine vector competence. Apoptosis has been shown to play a role in defense against viruses in insects and mammals. Although some observations suggest a correlation between apoptosis and resistance to arboviruses in mosquitoes, there is no direct evidence tying apoptosis to arbovirus vector competence. To determine whether apoptosis can influence arbovirus replication in mosquitoes, we manipulated apoptosis in Aedes aegypti mosquitoes by silencing the expression of genes that either positively or negatively regulate apoptosis. Silencing of the A. aegypti anti-apoptotic gene iap1 ( Aeiap1 ) caused apoptosis in midgut epithelium, alterations in midgut morphology, and 60 to 70% mosquito mortality. Mortality induced by Aeiap1 silencing was rescued by cosilencing the initiator caspase gene Aedronc , indicating that the mortality was due to apoptosis. When mosquitoes which had been injected with Aeiap1 double-stranded RNA (dsRNA) were orally infected with Sindbis virus (SINV), increased midgut infection and virus dissemination to other organs were observed. This increase in virus infection may have been due to the effects of widespread apoptosis on infection barriers or innate immunity. In contrast, silencing the expression of Aedronc , which would be expected to inhibit apoptosis, reduced SINV midgut infection and virus dissemination. Thus, our data suggest that some level of caspase activity and/or apoptosis may be necessary for efficient virus replication and dissemination in mosquitoes. This is the first study to directly test the roles of apoptosis and caspases in determining mosquito vector competence for arboviruses.

Background Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for human monocytic ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Methodology/Principal Findings Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Conclusions/Significance Morphological differences in the pathogen’s progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies in support of adaptation to vertebrate and tick cell environments.

Purpose To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS). Methods The anterior halves of whole eyeballs from chickens on embryonic (E) days E10 to E21 and Japanese quail on embryonic days E8 to E17 were fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence. Propidium iodide (PI) was used to detect the nuclei of osteocytes in scleral ossicles. FESEM and EDS were then used to show areas of mineralization and to identify differences in the elemental composition of different regions of the ossicles. Results Using Giemsa as a fluorescence stain, it was possible to observe the detailed morphology and development of both chicken and quail scleral ossicles. In chickens, bone microporosities first became visible at E15. Each microporosity contained a single nucleus, likely that of an osteocyte. The amount of carbon in ossicles steadily decreased during embryogenesis and post-hatching, while the concentration of oxygen showed a distinct increase over this time period. Calcium and phosphate levels in the ossicles increased gradually during embryonic and post-hatching stages. Conclusions A novel approach to study the development and mineralization of avian scleral ossicles during embryogenesis is presented. This methodology was validated by studying two different species, both important models for avian developmental research.

The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.

In most mammals, glucokinase (GK) acts as a hepatic “glucose sensor” that permits hepatic metabolism to respond appropriately to changes in plasma glucose concentrations. GK activity is potently regulated by the glucokinase regulatory protein (GKRP), which is encoded by the GCKR gene. GKRP binds GK in the nucleus and inhibits its activity. GK becomes active when it is released from GKRP and translocates to the cytosol. Low glucokinase (GK) activity is reported to be a principal feature of feline hepatic carbohydrate metabolism but the molecular pathways that regulate GK activity are not known. This study examined the hypothesis that species-specific differences in GKRP expression parallel the low GK activity observed in feline liver. Hepatic GKRP expression was examined using RT-PCR, immunoblot, and confocal immunomicroscopy. The results show that the GCKR gene is present in the feline genome but GCKR mRNA and the GKRP protein were absent in feline liver. The lack of GKRP expression in feline liver indicates that the low GK activity cannot be the result of GKRP-mediated inhibition of the GK enzyme. However, the absence of the permissive effects of GCKR expression on GK expression and activity may contribute to reduced GK enzyme activity in feline liver. The study results show that the cat is a natural model for GCKR knockout and may be useful to study regulation of GCKR expression and its role in hepatic glucose-sensing and carbohydrate metabolism.

Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis . We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis . The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

From studies using inhibitors such as tunicamycin and castanospermine, it has been suggested that plasma membrane glycoproteins may function as receptors in the phagocytosis of rod outer segments (ROS) by the retinal pigment epithelium (RPE). The exact structure of the oligosaccharide side chain of the glycoprotein may not be critical for this process. We have employed another inhibitor, swainsonine, which inhibits mannosidase II, a terminal enzyme in the protein glycosylation pathway, which results in membrane glycoproteins having hybrid-type oligosaccharide chains and fewer complex oligosaccharide chains. We have examined the ability of cultured rat RPE explants to phagocytize fluorescein isothiocyanate (FITC) labelled ROS or latex beads in the presence and absence of swainsonine. A significant (p less than 0.05) reduction in the phagocytosis of FITC-ROS was found between the swainsonine treated (37.7 +/- 4.1%) and untreated (85.4 +/- 2.7%) RPE explants. The nonspecific uptake of latex beads in both swainsonine treated (85.3 +/- 2.4%) and untreated (89.3 +/- 2.0%) RPE explants indicate that the RPE cells retained their ability to phagocytize. Major differences in spectrophotometric analysis of WGA-stained blots were an absence of a peak at 201 kD, a doublet at 86 kD and an overall reduction in all peak absorbances in the swainsonine treatments as compared to the untreated controls. These results suggest that the alterations in RPE glycoprotein formation due to swainsonine alter the ability of RPE to phagocytize ROS.

This study was conducted to improve the quality and theoretical understanding of gluten-free sorghum bread. The addition of 2% hydroxypropyl methylcellulose improved bread based on 105% water, 70% sorghum flour, and 30% potato starch. Nevertheless, a flat top and tendency toward a hole in the crumb remained. Sourdough fermentation of the total sorghum flour eliminated these problems. Size-exclusion high-performance liquid chromatography demonstrated that during sourdough fermentation, proteins from the dough liquid were degraded to peptides smaller than kafirin monomers (

Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells.Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy.RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize.The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.

Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress. The objectives of these studies are to determine how LEDGF controls PKC gamma activity in normal LECs: how this control of PKC gamma regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in LECs. A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF. PKC gamma protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng ml(-1). In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein-protein interaction between PKC gamma and Connexin 43. In the presence of LEDGF the activation of PKC gamma enhanced the phosphorylation of Connexin 43 by four-fold compared to the absence of LEDGF. The addition of LEDGF for 30 min resulted in a 65% decrease in gap junction Connexin 43 at the cell surface and a 70% decrease in gap junction activity. These results suggest that the activation of PKC gamma by LEDGF plays a major role in gap junction assembly/disassembly, which may enhance survival of LECs against osmolarity-stress induced by high sugar concentration.

PURPOSE. To determine the role of PKC in insulin-like growth factor (IGF)-I–induced phosphorylation of connexin (Cx)43 and control of gap junctions in lens epithelial cells. METHODS. N/N1003A rabbit lens epithelial cells were used in the experiments. PKC translocation or in vivo Cx43 phosphorylation on serine was determined by Western blot analysis. Gap junction activity was measured by scrape-loading/dye-transfer assay. The number of cell surface gap junction plaques was detected by confocal microscopy. The interaction between PKC and Cx43 was determined by coimmunoprecipitation. In vitro Cx43 phosphorylation was assayed by PKC assay kit. Endogenous sn-1,2-diacylglycerol (DAG) was measured by detecting 32 P-labeled phosphatidic acid. RESULTS. IGF-I stimulated activation and translocation of PKC in a dose- and time-dependent manner, acidic FGF (aFGF) had no effect on translocation of PKC, and PKC was not translocated by IGF-I at 25 ng/mL. PKC translocation resulted in coimmunoprecipitation with and phosphorylation of Cx43. IGF-I– or DAG-induced activation of PKC caused a decrease in gap junctions. IGF-I increased endogenous DAG. Exogenous CaCl2 and DAG stimulated PKC translocation. TMB-8, an internal calcium mobilization inhibitor, blocked CaCl2-induced PKC translocation; however, it had no effect on IGF-I– or DAG-induced translocation of PKC. CONCLUSIONS. PKC mediated IGF-I–induced decreases in gap junctional communication through interaction with and phosphorylation of Cx43. IGF-I caused an increase in DAG, and this increased translocation of PKC, whereas mobilization of calcium was not essential for IGF-I–stimulated translocation of PKC .( Invest Ophthalmol Vis Sci. 2003;44:1160 –1168) DOI

Little is known about the endocytosis and transcytosis of macromolecules into lens epithelium and fiber cells. The objective of this study was to determine if proteins (alpha-crystallins, beta-crystallins, and gamma-crystallins), carbohydrate (dextran), and plasmid DNA translocate from culture medium into these parts of the lens, with and without prior encapsulation into liposomes.alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were coupled with the fluorochrome Texas red, and plasmid DNA was labeled with propidium iodide. Adult rat lenses were incubated in medium containing one of these components with and without prior encapsulation of the macromolecule in commercially available liposomes (BioPORTER for alpha-crystallins, beta-crystallins, gamma-crystallins, and dextran; GenePORTER for plasmid DNA). Translocation of fluorescent macromolecule from the medium into the lens capsule, epithelium and fiber cells was monitored by confocal microscopy.alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were present in the capsule, epithelium, and fiber cells after 5 h of incubation. Translocation of fluorescent protein macromolecules into the epithelium was greatly facilitated by encapsulation in BioPORTER liposomes. These macromolecules were localized within the cytoplasm of epithelium and fiber cells. Plasmid DNA was localized to the epithelium, but not the fiber cells. Prior encapsulation of plasmid DNA into GenePORTER liposomes did not increase the intensity of fluorescence localized in epithelium. Without encapsulation, plasmid DNA preferentially localized to the nuclei of epithelial cells, while after encapsulation, plasmid DNA preferentially localized to the cytoplasm.After incubation with cultured lenses, large macromolecules comprised of proteins and carbohydrates were localized within the cytoplasm of epithelial cells and fiber cells. Prior encapsulation of protein macromolecules into BioPORTER liposomes facilitated the translocation of macromolecules into the cytoplasm of epithelium. Incubation of lenses with plasmid DNA resulted in localization to the epithelium, but not fiber cells. Localization of plasmid DNA was not facilitated by prior encapsulation in GenePORTER. Encapsulated DNA preferentially localized to the cytoplasm of epithelial cells, while without encapsulation, plasmid DNA localizes to the nuclei of epithelial cells. Together, these studies demonstrate that macromolecules of potential biological importance can readily pass through the lens capsule into epithelial cells and in some cases transcytose through the epithelium into fiber cells of the cortex. Furthermore, these studies suggest that prior encapsulation of protein macromolecules may be a possible therapeutic delivery system of physiologically important macromolecules into the epithelium and/or fiber cells of the intact lens.

Overexpression and activation of matrix metalloprotease (MMP) have been implicated in angiogenesis. However, the involvement of cysteine proteases, such as calpains (EC 34.22.17), is obscure. Thus, the purpose of this experiment was to study the involvement of cysteine proteases in angiogenesis induced by basic fibroblast growth factor (bFGF) in guinea pig and rat corneas using cysteine protease inhibitors. Sustained-release polymers containing bFGF were implanted into guinea pig and rat corneas to induce angiogenesis. For treatment of corneal angiogenesis, polymers containing cysteine protease inhibitors, leupeptin or SJA6017, were also implanted into corneas. Using the slit lamp, the corneas were observed for nine days after polymer implantation. Soluble proteins and albumin levels were used as markers of corneal injury by angiogenesis. bFGF induced angiogenesis in guinea pig and rat corneas. In guinea pig cornea, wet weight, the amount of soluble protein and albumin was highest at four days after bFGF-containing pellet implantation. In rat cornea, the amount of soluble protein and albumin was highest at six days, and wet weight increased within four days. One hundred nmole of leupeptin showed a tendency to reduce bFGF-induced angiogenesis in guinea pig cornea, and 10 nmole of SJA6017 was effective in reducing bFGF-induced angiogenesis in rat cornea, although SJA6017 showed a stronger effect than leupeptin. Ten nmole of SJA6017 significantly reduced the number of new blood vessels. These data suggested involvement of cysteine proteases in angiogenesis in guinea pig and rat cornea.

A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogenesis (selenite treated rats: galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.

To integrate past biochemical findings with past morphological observations of area insoluble material isolated from cataract and aged normal lenses, by determining the spatial distribution of alpha-crystallins associated with the plasma membrane (PM) of nuclear cataractous and age matched normal human lenses.Lenses were homogenized, pelleted and washed several times in 0.05M Tris-Cl (pH 7.2) containing 100mM KCl, 1 mM MgCl2 and 2mM beta-mercaptoethanol, followed by several washes in 8M urea. Urea insoluble pellets (UIP) were labeled before fixation and embedding with rabbit serum raised against alpha-crystallins, followed by goat anti-rabbit IgG conjugated to 5nm gold. Approximately 300 gold particles associated with the PM were counted, for each lens, on several electron microscopy (EM) micrographs. The number of gold particles/um of PM, number of individual vs clusters of gold particles were determined.Micrographs from both normal and cataractous human lenses clearly demonstrated the association of alpha-crystallins with the PM. Also apparent was the abundant labeling of the PM for cataractous lenses as compared to normal lenses. Quantification of the gold labeling revealed that not only was there an increase in the amount of labeling/um of PM in cataract lenses, but there was also an increased percentage of gold in clusters. These clusters were not only more numerous in cataractous lenses, but also contained a greater number of gold/cluster.These findings provide morphological evidence that the PM in nuclear cataract lenses is associated with large aggregates of alpha-crystallin.

Previous work by our laboratory has demonstrated that rod outer segment (ROS) phagocytosis can be mediated by mannose-receptor dependent activity. This study was designed to probe for potential ligands on the ROS surface which could interact with the mannose receptor during the phagocytic cycle. Solubilized ROS plasma membranes were passed over a mannose receptor-Sepharose column in the presence of CaCl2. Proteins specifically bound to the column were eluted using methyl-D-mannoside and EDTA and characterized by gel electrophoresis, lectin blots, and immunoblots. Silver stained gels of ROS plasma membrane proteins eluted from the mannose-receptor column demonstrated six bands: a major band at 36 kD, identified by monospecific antibodies as rhodopsin, and bands of Mr = 39 kD, 67 kD, 76 kD, 97 kD and 100 kD. Lectin blots of the eluted fractions confirmed that all six proteins in these fractions could bind concanavalin A. In summary, these results showed that rhodopsin and several other mannose-containing glycoproteins on ROS plasma membranes were bound to a mannose receptor column, and thus could serve as ligands for mannose receptor-mediated ROS phagocytosis.

Background One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.