Search

Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, but not HIF-2α protein: the salidroside-induced HIF-1α protein was via the reduction of HIF-1α degradation but not the mRNA induction. The increased HIF-1α could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Macromolecules present a remarkable potential as future therapeutics. However, their translation into clinical practice has been hampered by an inherently low bioavailability. Cell-penetrating peptides (CPP) have been recently shown to significantly improve on the bioavailability of macromolecules. Yet, the high cost associated with development and production of these peptides is a major factor hindering their rapid deployment beyond the laboratory. Here, we describe a facile and robust methodology for efficient and large-scale production of low-molecular-weight protamine-a potent CPP of great clinical potential. Our methodology is based on the immobilization of thermolysin, an enzyme catalyzing digestion of native protamine, on chemically surface-modified gels produced by silica sol-gel chemistry. Thermolysin was immobilized at extremely high matrix loading of 733 mg/g matrix and exhibited good thermal and pH stability, indicating robustness with respect to processing conditions. The mechanical properties of the silica matrix further allowed utilization of the immobilized thermolysin in both batch and packed-bed reactor systems to produce the LMWP peptide in high yields. Results presented here are of high significance as this efficient and cost-effective production of high purity LMWP could enable clinical translation of many potential macromolecular drugs., (Copyright © 2011 Wiley Periodicals, Inc.)

In sporadic Alzheimer's disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography-tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation.

The loss or dysfunction of the pancreatic endocrine β-cell results in diabetes. Recent innovative therapeutic approaches for diabetes aim to induce β-cell proliferation in vivo by pharmacological intervention. Based on the finding that overexpression of the transcription factor Nkx6.1 in islets in vitro increases β-cell proliferation while maintaining β-cell function, Nkx6.1 has been proposed as a potential target for diabetes therapy. However, it is unknown whether elevated Nkx6.1 levels in β-cells in vivo have similar effects as observed in isolated islets. To this end, we sought to investigate whether overexpression of Nkx6.1 in β-cells in vivo could increase β-cell mass and/or improve β-cell function in normal or β-cell-depleted mice. Using a bigenic inducible Cre-recombinase-based transgenic model, we analyzed the effects of Nkx6.1 overexpression on β-cell proliferation, β-cell mass, and glucose metabolism. We found that mice overexpressing Nkx6.1 in β-cells displayed similar β-cell proliferation rates and β-cell mass as control mice. Furthermore, after partial β-cell ablation, Nkx6.1 overexpression was not sufficient to induce β-cell regeneration under either nondiabetic or diabetic conditions. Together these results demonstrate that sustained Nkx6.1 overexpression in vivo does not stimulate β-cell proliferation, expand β-cell mass, or improve glucose metabolism in either normal or β-cell-depleted pancreata. Thus, raising cellular Nkx6.1 levels in β-cells in vivo is unlikely to have a positive impact on type 2 diabetes.

The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the "bait" protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

The past 20 years of experience with umbilical cord blood transplantation have demonstrated that cord blood is effective in the treatment of a spectrum of diseases, including hematological malignancies, bone marrow failure, hemoglobinopathies, and inborn errors of metabolism. Cord blood can be obtained with ease and then safely cryopreserved for either public or private use without loss of viability. As compared to other unrelated donor cell sources, cord blood transplantation allows for greater human leukocyte antigen disparity without a corresponding increase in graft-vs.-host disease. Moreover, cord blood has a lower risk of transmitting infections by latent viruses and is less likely to carry somatic mutations than other adult cells. Recently, multiple populations of stem cells with primitive stem cell properties have been identified from cord blood. Meanwhile, there is an increasing interest in applying cord blood mononuclear cells or enriched stem cell populations to regenerative therapies. Accumulating evidence has suggested functional improvements after cord blood transplantation in various animal models for treatments of cardiac infarction, diabetes, neurological diseases, etc. In this review, we will summarize the most recent updates on clinical applications of cord blood transplantation and the promises and limitations of cell-based therapies for tissue repair and regeneration., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

This study was aimed to investigate the expression level of NOV and BNIP3 mRNA in mice myelomonocytic leukemia (AML-M(4)) and its significance. The mice were inoculated intravenously with myelomonocytic leukemia cells of WEHI-3, and divided randomly into chemotherapy group and control (untreated) group. Bone marrow samples were then collected from both groups at different times. The NOV and BNIP3 mRNA expression were detected by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and the relationship between these expression levels and clinical significance in leukemia incidence and progression were analyzed with β-actin as the housekeeping gene. The results showed that the mean values of NOV and BNIP3 increased gradually from 2 weeks after inoculation and achieved highest level at death in control group. Expression level of NOV increased from 1.85E-05 before inoculation to 3.57E-02 at death (p < 0.05), and BNIP3 from 3.44E-03 to 3.48E-02. While 2 gene expression in the chemotherapy group decreased quickly to 2.51E-05 and 1.58E-03 (p < 0.05) respectively after chemotherapy, which were close to the level before inoculation (p > 0.05). The 2 gene expressions again rose at relapse, and difference of expression level between 2 group at death were no statistically significant (p > 0.05). It is concluded that the expression of NOV and BNIP3 in leukemia AML-M(4) is significantly higher than that in normal controls, of which high level expression is an important factor in the development of leukemia. Close relation between the therapeutic effect and expression level of these two genes suggests the great value in prognostic evaluation and MRD detection.

While biocatalysts show tremendous potential for the industrial production of fine chemicals, their integration into large-scale processes has been slow. One of the main reasons for slow acceptance in industry is the inherent instability of the enzymes. Recent developments in bioengineering have shed some light on methods of improving enzyme stability. One method that has been used for many decades, successfully to varying degrees, has been the immobilization of enzymes. To this regards, silica gels have attracted much attention because of the ease of surface functionalization, high surface areas, mechanical and thermal stability, and resistance to both chemical and biological attack. We have previously shown the immobilization of invertase on silica gels with high immobilized activity and significantly improved stability. Here, we provide greater details on the methods for effecting the immobilization.

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.

Andrographolide (Ap) is a bioactive compound in Andrographis paniculata that is a Chinese herb. The pi class of glutathione S-transferase (GSTP) is one kind of phase II detoxification enzyme. Here we show that induction of GSTP protein and mRNA expression in rat primary hepatocytes by Ap was inhibited by forskolin and a variety of cAMP analogues. The inhibitory effect of the cAMP analogues was partially blocked by pretreatment with H89. In the presence of Ap, forskolin, or both, the expression of phospho-cAMP response element-binding protein (CREB) was increased. Ap alone had no effect on inducible cAMP early repressor (ICER) mRNA expression; however, Ap played a potentiating role in forskolin-induced ICER mRNA expression. An EMSA and immunoprecipitation assay showed that ICER binding to cAMP-response element (CRE) was increased in cells cotreated with Ap and forskolin for 3 and 8 h. Taken together, these results suggest that ICER is likely to be involved in the suppression of Ap-induced GSTP expression caused by the increase of cAMP in rat primary hepatocytes.

Motivation: An important application of gene expression microarray data is the classification of samples into categories. Accurate classification depends upon the method used to identify the most relevant genes. Owing to the large number of genes and relatively small sample size, the selection process can be unstable. Modification of existing methods for achieving better analysis of microarray data is needed., Results: We propose a Bayesian stochastic variable selection approach for gene selection based on a probit regression model with a generalized singular g-prior distribution for regression coefficients. Using simulation-based Markov chain Monte Carlo methods for simulating parameters from the posterior distribution, an efficient and dependable algorithm is implemented. It is also shown that this algorithm is robust to the choices of initial values, and produces posterior probabilities of related genes for biological interpretation. The performance of the proposed approach is compared with other popular methods in gene selection and classification via the well-known colon cancer and leukemia datasets in microarray literature., Availability: A free Matlab code to perform gene selection is available at http://www.sta.cuhk.edu.hk/xysong/geneselection/., Supplementary Information: Supplementary data are available at Bioinformatics online.

Objective: Lidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses., Methods: Cleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively., Results: SIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin., Conclusions: Cellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.

Posttranslational modifications such as phosphorylation and ubiquitination serve, independently or together, as gatekeepers of protein transport and turnover in normal and disease physiologies. Aberrant protein phosphorylation is one of the defining pathological hallmarks of more than 20 different neurodegenerative disorders, including Alzheimer's disease (AD). The disruption of the phosphorylation of neurotransmitter receptors has been implicated as one of the causal factors of impaired memory function in AD (1-3). Another feature of AD is the aberrant accumulation of proteins that are normally degraded by the ubiquitin proteasome system upon being conjugated to ubiquitin. Thus, elucidating the protein targets of phosphorylation and ubiquitination that can serve as AD biomarkers will aid in the development of effective therapeutic approaches to the treatment of AD. This chapter provides details pertaining to the qualitative and quantitative liquid chromatography tandem mass spectrometry-based analysis of an affinity purified, phosphorylated, and ubiquitinated protein, paired-helical filament tau.

Andrographis paniculata (Ap) is a commonly used herb for traditional medicine in many Southeast Asian countries. In the present study, we investigated the effect of Ap on the expression of the pi class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 25 or 50 microg/mL of ethanol or ethyl acetate extracts of Ap (ApEE or ApEAE) or 10 or 20 microM andrographolide, which is the major active diterpene lactone of Ap, for 48 h. ApEE, ApEAE, and andrographolide dose-dependently induced GSTP protein and mRNA expression. In a GST activity assay, GST activity was significantly higher in cells treated with the maximum concentrations of ApEE, ApEAE, and andrographolide than in control cells (P<0.05). The pTA-2713 luciferase reporter construct containing rat GSTP enhancer 1 (GPE1) was transiently transfected into Clone 9 liver cells. Cells treated with ApEE, ApEAE, and andrographolide showed a dose-dependent increase in luciferase activity. GPE1 deletion abolished the induction efficiency of Ap. Also, the induction of GSTP expression by Ap was inhibited by wortmannin, which is an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. These results indicate that ApEE, ApEAE, and andrographolide induce GSTP expression. This induction is likely related to the PI3K/Akt pathway, and GPE1, an enhancer element in GSTP promoter, is essential for the induction.

In this paper, we perform surface plasmon-coupled emission studies on Rhodamine 6G molecules embedded in a corrugated structure of a thin film composed of fluorinated silica particles, and a binding medium. Our results show enhancements of photoluminescence due to surface corrugation. By varying the size of the fluorinated silica nanoparticles we were able to control the surface correlation length scale of the corrugated surface structure. It was found that the coupling efficiency of the directional light emission is strongly correlated to the surface morphology, particularly the surface correlation length, of the corrugated dielectric structure. This substantial enhancement of signal could potentially be utilized in Organic Light Emitting Diode devices to enhance the light emission and transmission through a thin silver layer which can also serve as the cathode in Top-Emitting Organic Light Emitting Diodes.

Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases.

Seventy-two isolates of carbapenem-resistant Pseudomonas aeruginosa (CRPA) collected through the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program in 2000 and 2002 were studied for carriage of metallo-beta-lactamase (MBL), integrase genes, and integrons. Epidemiologic relatedness was determined using pulsed-field gel electrophoresis. The prevalence of the MBL genes among CRPA was 36.0% (9 of 25) in TSAR II and 17.0% (8/47) in TSAR III, with prevalence in surveyed hospitals being 19.1% (4/21, TSAR II) and 23.1% (6 of 26, TSAR III). The bla(VIM-3) was detected in 15 of the 17 MBL-positive isolates; the remainder possessed bla(VIM-2). The bla(IMP) was not evident. Class 1 integron was detected in all MBL-positive isolates; amplicon DNA sequences containing the bla(VIM-3) regions were all identical. All MBL-positive isolates remained susceptible to colistin. Molecular typing revealed a predominant strain that comprised 14 of the isolates among the various surveyed hospitals.

Background & Objective: Vasculogenic mimicry (VM) is a blood supply pattern in malignant tumors. Mimic vessel walls are formed by tumor cells, not by endothelial cells. Previous studies of VM are limited on malignant tumors with high aggression and bi-directional differentiation potentials, but seldom on epithelium-originated tumors. This study was to observe VM in hepatocellular carcinoma (HCC) by 3-dimensional culture of HCC cell line HepG2 and immunohistochemistry., Methods: Three-dimensional culture system of HepG2 cells was constructed to observe VM. The presence of VM in 15 specimens of HCC was observed by immunohistochemical and histological double staining of CD31 and PAS, or Ferritin and PAS., Results: During 3-dimensional culture, HepG2 cells stretched out thin and long apophyses at the second day, and linked each other to form wreath and net-work structure at the seventh day. All of the 15 HCC simples showed CD31-positive for endothelial cells, and Ferritin-positive for tumor cells. The immunohistochemical and histological double staining also proved that VM, formed by CD31-negative and Ferritin-positive tumor cells, existed in 7 of the 15 simples of HCC. Tumor cells were separated from the tubes by PAS-positive matter like basement membrane. Red blood cells could be seen in the tubes. The number of VM was less in well differentiated simples than in poorly differentiated samples; CD31-positive tumor cells were observed in poorly differentiated samples., Conclusions: HepG2 cells have the capacity of self-metamorphosing and vasculorizing. Tumor cells can obtain oxygen and nutrition through VM.

Objective: To explore effect of acupuncture at Shaoze (SI 1) for treatment of postpartum hypolactation., Methods: Multicentral randomized controlled and single blind clinical trial was adopted, and 276 cases were divided into a treatment group and a control group, 138 cases in each group. The treatment group were treated with electroacupuncture (EA) at Shaoze (SI 1) and the control group with EA at Shangyang (LI 1). After treatment of 2 courses, the therapeutic effects and changes of cumulative score of TCM symptoms, mammary filling degree, lactation amount, prolactin level were evaluated and investigated., Results: The cured and markedly effective rate was 97.8% in the treatment group and 24.3% in the control group with a significant difference between the two groups (P < 0.05). The treatment group in improvement of the cumulative score of TCM symptoms and the mammary filling degree, and increasing the lactation amount and the prolactin level were better than the control group (P < 0.01)., Conclusion: Acupuncture at Shaoze (SI 1) has obvious therapeutic effect on hypolactation.

The role of post-translational modification, such as sumoylation, in modulating the efficacy of doxorubicin (Dox) treatment remains unclear. Transcriptional cofactor KRAB domain-associated protein 1 (KAP1) has been shown to complex with the KRAB zinc finger protein, ZBRK1, to repress the transcription of target genes. Through a combination of proteomic screening and site-directed mutagenesis approaches, we have identified lysines 554, 779, and 804 as the major sumoylation sites in KAP1. We then present evidence that Dox-mediated induction of cell cycle regulator p21 expression is differentially regulated by KAP1 sumoylation status. Moreover, the KAP1 sumoylation level was transiently decreased upon Dox exposure, and transfection with the KAP1 sumoylation mimetic, SUMO-1-KAP1, desensitizes breast cancer MCF-7 cells to Dox-elicited cell death. The sumoylation-dependent stimulation of KAP1 function is achieved by enhancing the methylation of H3-K9 and attenuating the acetylation of H3-K9 and H3-K14 at the p21 core promoter. We also show that occupancy of ZBRK1 response elements located at the p21 promoter by ZBRK1.KAP1 is independent of KAP1 sumoylation. Hence, sumoylation of KAP1 represses p21 transcription via a chromatin-silencing process without affecting interaction between KAP1.ZBRK1 and DNA, thus providing a novel mechanistic basis for the understanding of Dox-induced de-repression of p21 transcription. Taken together, our results suggest that Dox-induced decrease in KAP1 sumoylation is essential for Dox to induce p21 expression and subsequent cell growth inhibition in MCF-7 cells.

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.

One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of paired helical filaments (PHFs) of hyperphosphorylated microtubule-associated protein Tau. Tandem mass spectrometry was employed to examine PHF-Tau post-translational modifications, in particular protein phosphorylation and ubiquitination, to shed light on their role in the early stages of Alzheimer disease. PHF-Tau from Alzheimer disease brain was affinity-purified by MC1 monoclonal antibody to isolate a soluble fraction of PHF-Tau in a conformation unique to human AD brain. A large number of phosphorylation sites were identified by employing a data-dependent neutral loss algorithm to trigger MS3 scans of phosphopeptides. It was found that soluble PHF-Tau is ubiquitinated at its microtubule-binding domain at residues Lys-254, Lys-311, and Lys-353, suggesting that ubiquitination of PHF-Tau may be an earlier pathological event than previously thought and that ubiquitination could play a regulatory role in modulating the integrity of microtubules during the course of AD. Tandem mass spectrometry data for ubiquitin itself indicate that PHF-Tau is modified by three polyubiquitin linkages, at Lys-6, Lys-11, and Lys-48. Relative quantitative analysis indicates that Lys-48-linked polyubiquitination is the primary form of polyubiquitination with a minor portion of ubiquitin linked at Lys-6 and Lys-11. Because modification by Lys-48-linked polyubiquitin chains is known to serve as the essential means of targeting proteins for degradation by the ubiquitin-proteasome system, and it has been reported that modification at Lys-6 inhibits ubiquitin-dependent protein degradation, a failure of the ubiquitin-proteasome system could play a role in initiating the formation of degradation-resistant PHF tangles.

Increasing evidence suggests that oxidative injury is involved in the pathogenesis of many age-related neurodegenerative disorders, including Alzheimer's disease (AD). Identifying the protein targets of oxidative stress is critical to determine which proteins may be responsible for the neuronal impairments and subsequent cell death that occurs in AD. In this study, we have applied a high-throughput shotgun proteomic approach to identify the targets of protein carbonylation in both aged and PS1 + AbetaPP transgenic mice. However, because of the inherent difficulties associated with proteomic database searching algorithms, several newly developed bioinformatic tools were implemented to ascertain a probability-based discernment between correct protein assignments and false identifications to improve the accuracy of protein identification. Assigning a probability to each identified peptide/protein allows one to objectively monitor the expression and relative abundance of particular proteins from diverse samples, including tissue from transgenic mice of mixed genetic backgrounds. This robust bioinformatic approach also permits the comparison of proteomic data generated by different laboratories since it is instrument- and database-independent. Applying these statistical models to our initial studies, we detected a total of 117 oxidatively modified (carbonylated) proteins, 59 of which were specifically associated with PS1 + AbetaPP mice. Pathways and network component analyses suggest that there are three major protein networks that could be potentially altered in PS1 + AbetaPP mice as a result of oxidative modifications. These pathways are 1) iNOS-integrin signaling pathway, 2) CRE/CBP transcription regulation and 3) rab-lyst vesicular trafficking. We believe the results of these studies will help establish an initial AD database of oxidatively modified proteins and provide a foundation for the design of future hypothesis driven research in the areas of aging and neurodegeneration.

Objective: We established diet-induced obesity animal model and observed the influence of obesity on testicle of the male rats., Methods: Rats in model group were fed with fat-enriched diet for 6 weeks. We measured the body weight and the testis weight and observed the development of testicle by microscope, and compared with those of control group. Meanwhile, we measured the serum levels of testosterone (T) and estradiol (E2)., Results: The average body weight in model group rats was higher 29% than that in control group at 6 weeks old, 30% at 9 weeks old. We observed under microscope that most of the convoluted seminiferous tubule developed no good in model group, the 4 layer cells was sparse and the arrangement of them was not in order. The testosterone to estradiol ratios(T/E2) in model group was significant lower then that of control group (P < 0.05)., Conclusion: The fat-enriched fat diet can induce obesity. It influenced the development of the testicle. This probably related to decrease of the testosterone to estradiol ratios.

Abstract Oxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimer's disease (AD). Given that amyloid beta (Abeta) itself generates ROS that can directly damage proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Abeta-mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two-dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Abeta-induced oxidative stress in cultured primary cortical mouse neurons. The Golgi-resident enzyme glucuronyltransferase (GlcAT-P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK-1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Abeta, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK-1 expression. Moreover, in vivo, HNK-1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Abeta-mediated oxidation of GlcAT-P is impairment of its enzymatic function, thereby disrupting HNK-1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK-1 modulates synaptic plasticity and is critically involved in memory consolidation.

Purpose: Amyloid-beta (Abeta) is a self-aggregating protein found in senile plaques in Alzheimer's disease (AD) brain and is thought to play a major role in the disease process. Oxidative stress may be a predominant cause of the formation of these Abeta aggregates. This study aims at identifying possible sites of copper-catalyzed oxidation of Abeta1-40 using liquid chromatography tandem mass spectrometry (LC/MS/MS) and scoring algorithm for spectral analysis (SALSA). Traditionally, identification of post-translational modifications by tandem mass spectrometric analysis requires users to inspect manually thousands of MS/MS spectra, which can be a tedious and time-consuming process. With the use of SALSA, users can automatically search for post-translational modifications based on the spacing of the m/z values associated with the ion series of an amino acid sequence., Methods: Abeta1-40 was subjected to copper-catalyzed oxidative stress. LC/MS/MS and SALSA analyses were used to determine the sites of post-translational modification within the tryptic fragments., Results: Oxidation was found to occur preferentially at the histidine residues Hisl3 and Hisl4 and at the methionine residue (Met35) of Abeta1-40., Conclusions: The combination of LC/MS/MS and SALSA searches could dramatically improve the efficiency and accuracy of determining the specific sites of oxidation of in vitro, copper-oxidized Abeta1-40 as well as other oxidized proteins.

Alterations in the trafficking and function of the endocytic pathway have been extensively documented to be one of the earliest pathological changes in sporadic Alzheimer's disease (AD). Although the pathophysiological consequences of these endosomal/lysosomal changes are currently unknown, several recent studies have suggested that such changes in endocytic function are able to cause a redistribution of several lysosomal hydrolases into early endosomes, leading to the overproduction of neurotoxic amyloid peptide. Recently, we and others have demonstrated that abnormal endocytic pathology within post-mitotic neurons can, in part, be attributed to alterations in sphingomyelin/ceramide metabolism, resulting in the intracellular accumulation of ceramide. Once inside the cell, the ability of ceramide to physically alter membrane structure, formation, and fusion, rather than serving solely as a lipid secondary messenger, may severely compromise normal endocytic trafficking. In this review, we will discuss the potential pathological effects of abnormal sphingomyelin/ceramide metabolism on intracellular vesicular transport in relation to both amyloid accumulation in AD and various neurodegenerative diseases associated with lysosomal abnormalities.

Purpose: The major initiative of this study was to implement a novel proteomic approach in order to detect protein carbonylation in aged mouse brain. Several lines of evidence indicate that reactive oxygen species (ROS)-induced protein oxidation plays an essential role in the initiation of age-related neuropathologies. Therefore, the identification of free radical or peroxide substrates would provide further insight into key biochemical mechanisms that contribute to the progression of certain neurological disorders., Methods: Historically, ROS targets have been identified by conventional immunological two-dimensional (2-D) gel electrophoresis and mass spectrometric analyses. However, specific classes of proteins, such as transmembrane-spanning proteins, high-molecular-weight proteins, and very acidic or basic proteins, are frequently excluded or underrepresented by these analyses. In order to fill this technologic gap, we have used a functional proteomics approach using a liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis coupled with a hydrazide biotin-streptavidin methodology in order to identify protein carbonylation in aged mice., Results: Our initial studies suggest an ability to identify at least 100 carbonylated proteins in a single LC-MS/MS experiment. In addition to high-abundance cytosolic proteins that have been previously identified by 2-D gel electrophoresis and mass spectrometric analyses, we are able to identify several low-abundance receptor proteins, mitochondrial proteins involved in glucose and energy metabolism, as well as a series of receptors and tyrosine phosphatases known to be associated with insulin and insulin-like growth factor metabolism and cell-signaling pathways., Conclusions: Here we describe a rapid and sensitive proteomic analysis for the identification of carbonylated proteins in mouse brain homogenates through the conjunction of liquid chromatography and tandem mass spectrometry methods. We believe the ability to detect these post-translationally modified proteins specifically associated with brain impairments during the course of aging should allow one to more closely and objectively monitor the efficacy of various clinical treatments. In addition, the discovery of these unique brain biomarkers could also provide a conceptual framework for the future design of alternative drugs in the treatment of a variety of age-related neurodegenerative disorders.

Impairment of endosomal/lysosomal functions are reported as some of the earliest changes in several age-related neurological disorders such as Alzheimer's disease. Dysregulation of the lysosomal system is also accompanied by the accumulation of age-associated pigments and several recent reports have indicated that this age-related lipofuscin accumulation can sensitize cells to oxidative stress and apoptotic cell death. In this study, we have established and evaluated an in vitro age-related pathology paradigm that models lipofuscin accumulation. Our model consists of the treatment of cultured primary mouse neurons with lysosomotropic detergents. We have observed that one of the earliest biochemical changes associated with lysosomotropic detergent-induced membrane instability is a loss of the endosomal/lysosomal proton gradient integrity, followed by an activation of sphingomyelin hydrolysis and ceramide accumulation within enlarged endosomal/lysosomal vesicles. In addition, we demonstrate that ceramide accumulation correlates with the activation of proximal procaspases-8 and -9 as well as distal caspase-3, prior to the appearance of cell death. Taken together, we propose that disturbances of the endosomal/lysosomal system, in addition to the activation of the sphingomyelinase hydrolysis cycle, play essential roles in the course of post-mitotic neuronal aging. The abnormal accumulation of undigested lipids and proteins within dysfunctional endosomal/lysosomal vesicle populations during the process of pathological aging may serve as triggers of the cell death programs that are associated with downstream neurodegeneration.

To provide an explanation for earlier paradoxical findings of lithium on survival of mature and immature neurons, this study monitors changes in cytosolic caspases in rat cerebellar granule cells (CGC) grown 2-7 days in vitro (DIV), or in murine E-17 cortical neurons. Data show Li+ protects mature 7-DIV CGC parallel to a decrease in proximal and distal caspases but increases levels for immature 2-DIV-CGC or E-17 cortical neurons. Caspases mirror viability based on morphological analyses (dye uptake, phase-contrast, DNA fragmentation), and suggest protection occurs by suppressing activation of a cascade resulting in distal effectors that destroy proteins essential for neuronal survival. Protection was dose-dependent with EC50 3.0 mM and extended to 64 h in K+-serum deprived apoptotic media. Neuronal extracts contain a spectrum of proximal (-2, -8, -9) and distal (-3, -6) caspases sensitive to Li+ on assay with preferred peptide substrates and by immunoblotting. The lack of direct effect on activated cytosols indicates Li+ acts upstream only on intact cells, at sites for recruitment of pivotal procaspases. Alterations of procaspase-9 p46 and membrane-bound cytochrome c (Apaf-1) point to interaction with an intrinsic Mt-mediated pathway as one of the targets. The opposite effects on caspases and viability of immature or embryological neurons point to existence of alternative pathways that alter during neurite outgrowth suggesting the use of Li+ as a probe to unravel events relevant to neurogenesis.

Our previous studies suggest that a failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes is a mechanism that contributes to intracellular accumulation in Alzheimer's disease. In this study, we demonstrate that cultured primary neurons are able to internalize soluble Abeta1-42 from the culture medium and accumulate inside the endosomal/lysosomal system. The intracellular Abeta1-42 is resistant to protease degradation and stable for at least 48 h within the cultured neurons. Incubation of cultured neurons with a cytotoxic concentration of soluble Abeta1-42 invokes the rapid free radical generation within lysosomes and disruption of lysosomal membrane proton gradient which precedes cell death. The loss of lysosomal membrane impermeability is only specific to the Abeta1-42 isoform since incubation of cells with high concentrations of Abeta1-40 has no effect on lysosomal hydrolase release. To further support the role of lysosomal membrane damage in Abeta-mediated cell death, we demonstrate that photodisruption of acridine orange (AO)-loaded lysosomes with intense blue light induces a relatively rapid synchronous lysosomal membrane damage and neuronal death similar to that observed as a result of Abeta exposure. AO leaks quickly from late endosomes and lysosomes and partially shifts the fluorescence from an orange fluorescence to a diffuse, green cytoplasmic fluorescence. Such AO relocalization is due to an initial disruption of the lysosomal proton gradient, followed by the release of lysosomal hydrolases into the cytoplasmic compartment. Treatment of cells with either the antioxidant n-propyl gallate or lysosomotropic amine (methylamine) partially blocks the release of lysosomal contents suggesting that this AO relocalization is due to lysosomal membrane oxidation. Based on these findings, we propose that the cell death mediated by the soluble Abeta may be fundamentally different from the cell loss observed following extracellular Abeta deposition., (Copyright 2001 Academic Press.)

Recent studies showing that microglia internalize the amyloid beta-peptide (Abeta) suggest that these cells have the potential for clearing Abeta deposits in Alzheimer's disease, and mechanisms that regulate the removal of Abeta may therefore be of clinical interest. Previous studies from this laboratory showing that C1q enhances phagocytosis of cellular targets by rat microglia prompted the current investigations characterizing the effects of C1q on microglial phagocytosis of Abeta. Microglia were shown to phagocytose Abeta1-42, in agreement with observations of other investigators. Uptake of Abeta1-42 was observed for concentrations of 5-50 microM, and phagocytosis of peptides containing (14)C or fluorescein (FM) labels was not affected by the interaction of microglia with C1q-coated surfaces. However, inclusion of C1q (125 nM-1.4 microM) in solutions of 50 microM Abeta1-42 inhibited the uptake of (14)C-Abeta1-42 and FM-Abeta1-42, suggesting that C1q blocks the interaction of Abeta with microglia. Uptake of Abeta was partially blocked by the scavenger receptor ligands polyinosinic acid and maleylated BSA. Inhibition of Abeta uptake by C1q may contribute to the accumulation of fibrillar, C1q-containing plaques that occurs in parallel with disease progression. These data suggest that mechanisms which interfere with the binding of C1q to Abeta may be of therapeutic value both through inhibition of the inflammatory events resulting from complement activation and via altered access of Abeta sites necessary for ingestion by microglia., (Copyright 2000 Academic Press.)

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.

A critical issue concerning Alzheimer's disease is its selectivity, which leads to cellular degeneration in certain brain areas but not in others, and whether this pathogenic selectivity involves products of the amyloid precursor protein (APP). Here, we show that the amyloid beta protein Abeta1-42 is accumulated gradually and is retained intact by field CA1, but not by other subdivisions, of organotypic hippocampal slice cultures. In contrast, the slightly shorter Abeta1-40 peptide was not sequestered selectively. Sequestration of Abeta1-42 was followed by the build-up of carboxyterminal fragments of the endogenous precursor protein that were identified by immunoprecipitation. Unlike the peptide uptake, this induction appeared to be stochastic at the cellular level. In addition, the APP fragments were distributed more broadly within the CA1 pyramidal neurons than the sequestered Abeta1-42, and they appeared to be localized to synaptic terminals in the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of the subfield CA3. Concentrations of synaptophysin, a presynaptic marker, decreased as the number of neurons producing amyloidogenic species increased. These results indicate that exogenous Abeta1-42 sets into motion a sequence that involves 1) selective uptake of the peptide by vulnerable cells at risk in Alzheimer's disease, 2) markedly enhanced production of amyloidogenic precursor material, and 3) slow deterioration of central synapses.

Previous studies have implicated the failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes as a mechanism for the accumulation of beta-amyloid in Alzheimer's disease. We examined the consequences of intracellular accumulation of Abeta1-42 on the integrity of the endosomal/lysosomal compartment by monitoring Lucifer Yellow fluorescence and the release of lysosomal hydrolases into the soluble, cytosolic fraction. In control cells, the Lucifer Yellow fluorescence is observed as punctate staining in a perinuclear distribution with no apparent cytoplasmic fluorescence, consistent with its localization in late endosomes or secondary lysosomes. After incubation with Abeta1-42 for 6 hr, a loss of lysosomal membrane impermeability is observed as evidenced by redistribution of the fluorescence to a diffuse, cytoplasmic pattern. The loss of lysosomal membrane impermeability is correlated with Abeta1-42 accumulation, since incubation of the cells with the nonaccumulating isoform of amyloid, Abeta1-40, does not induce leakage. The same results were obtained using the release of soluble lysosomal hydrolases, cathepsin D and beta-hexosaminidase, into the cytosol as an assay for the leakage of lysosomal contents. Together, our results suggest that the loss of lysosomal membrane impermeability may be an early event in Abeta pathogenesis, and provide an explanation for the miscompartmentalization of extracellular and cytoplasmic components observed in Alzheimer's disease (AD). The release of hydrolases may further cause the breakdown of the cytoskeleton and the blebbing of the plasma membrane, and the leakage of heparan sulfate glycosaminoglycans from the lysosome may ultimately promote the assembly of tau into neurofibrillary tangles (NFT).

The identification of the cellular and subcellular regions of the Alzheimer's disease brain to which the presenilin-1 (PS-1) protein localizes is expected to contribute to an understanding of its pathophysiological role. Toward this end, we have derived an affinity-purified antibody to a synthetic PS-1 peptide. In this report, we demonstrate that this antibody, called SW2, specifically recognizes full-length, 47-kDa PS-1 protein from rat primary cortical neurons, from a human neuronal cell line, and from human brain extracts on Western immunoblots. Immunohistochemical analysis of postmortem brain tissue from control and Alzheimer's disease patients using this SW2 antibody indicates an intracellular localization of PS-1 immunoreactivity with prominent perinuclear characteristics in neurons, with staining also detected in neuritic processes. Despite various treatments of the tissue sections, no PS-1 immunoreactivity was observed in neuritic plaques, the hallmark pathological lesions of Alzheimer's disease. In addition, confocal microscopic analysis of immunostained cultured primary neurons revealed a prominent perinuclear pattern of PS-1 immunoreactivity consistent with vesicular localization, as well as punctate staining in neuritic processes.

Escherichia coli has a ribonucleoprotein complex that is composed of a 114 nucleotide 4.5S RNA and a 48 kDa polypeptide (P48) that has been demonstrated to function in translation and in the secretion of periplasmic polypeptides. A small RNA of approximately 220 nucleotides has been identified in maize mitochondria that includes sequence identity with the highly conserved domain of the bacterial 4.5S RNA. The transcript is mitochondrially encoded and maps to a region upstream of the gene for ATP synthase subunit I. The mitochondrial 4.5S-like RNA has 15 nucleotides of sequence identity with the highly conserved region of the bacterial 4.5S RNA. Sucrose density gradient centrifugation of a maize mitochondrial lysate demonstrated that the 4.5S RNA is a component of a high molecular weight complex under native conditions, and could be disrupted by phenol. Anti-P48 immune serum immuno-precipitated a mitochondrial protein of approximately 48 kDa, and RNA gel blot analysis of the immunoprecipitation reaction indicated that the 4.5S-like RNA co-immuno-precipitated with the 48 kDa polypeptide. The mitochondrial 4.5S ribonucleoprotein complex could function in translation or protein targeting.

We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.

Boehm and Martire have shown that the Flory-Huggins (FH) lattice model applied to mixtures of squares and rigid rods in solvent on a two dimensional lattice gives different results depending on whether rods or squares are placed first onto the lattice. This correct derivation places the validity of the FH model itself into question since the final result should be independent of the order of placement. An analysis of the FH model in terms of Poisson statistics suggests an alternative formula for the probability of successfully placing a rectangle into an area partially filled with other rectangles, which when incorporated into the FH counting procedure gives the exact thermodynamic result for the tiling of squares (i.e., no solvent and no rods). An attempt to solve the order of placement problem is made by solving the problem of one square plus any number of rods and then generalizing the statistics so that they are consistent with this result. Equations are given for squares plus rods plus solvent in both two and three dimensions. For plates plus solvent in three dimensions a purely entropy driven phase transition between an anisotropic layered phase and an isotropic phase is obtained. This transition is analogous to the isotropic to nematic liquid crystal phase transition in rigid rods. Our equations, when augmented by energy considerations, are useful for calculating the equilibrium properties of discotic systems, polymer-layered silicate composites, and the adsorption of plate like molecules onto surfaces.

Immobilization of protamine to the inner lumen of cellulose hollow fibers has been shown useful in preventing both heparin- and protamine-induced complications during an extracorporeal blood circulation procedure. The current study examined the effects of variables on the immobilization of protamine to cyanogen bromide (CNBr)-activated cellulose hollow fibers. The degree of protamine immobilization was controlled by three independent parameters: the amount of CNBr used during the activation process, the duration of the coupling process, and the protamine concentration in the coupling solution. By the adjustment of these parameters, cellulose fibers containing desired amounts of immobilized protamine (ranging from 1 to 20 mg of immobilized protamine per gram of dry fibers) were readily prepared.Heparin adsorption to the protamine-bound cellulose fibers was also examined. The adsorption isotherm followed a Langmuir adsorption model. The amount of heparin adsorbed was dependent on both the heparin concentration in the substrate solution and the protamine loading on the fibers. The Langmuir adsorption constant K was estimated to be 0.37 +/- 0.06 mL/mg, whereas the saturation capacity Q(s) of the protamine-bound fibers increased with increasing the protamine loading.

Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.