Augmentation of specific tumor immunity against a syngeneic SV40-induced sarcoma in mice by retinoic acid.

The effects of retinoic acid, a vitamin A derivative, which has been shown to have immune adjuvant properties, were studied in vivo in a syngeneic tumor system by the use of the tumor-cell neutralization assay. The effector activity of spleen cells of BALB/c mice immune against a syngeneic SV40-induced sarcoma, mKSA, was specifically augmented by low doses of retinoic acid, whereas high doses had a suppressive effect. In addition, the time required for generation of the effector activity was shortened and the immune activity lasted longer in the retinoic acid-treated mice. With the use of cell-depletion techniques it was demonstrated that thymus-derived lymphocytes were affected by retinoic acid.     

Studies on the mechanism of action of hexamethylene bisacetamide, a potent inducer of erythroleukemic differentiation

Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up by the cell; an intracellular concentration equal to the extracellular concentration is achieved by 6–8 h. Commitment to differentiation is not detected until at least 10 h after equilibration. Both uptake and commitment to differentiate are concentration and temperature dependent. The majority of the compound is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in both cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, but acetylation of the diamine greatly increases inducing activity.     

Labeling of soybean agglutinin by oxidation with sodium periodate followed by reduction with sodium [3-H]borohydride.

Periodate oxidation of soybean agglutinin, a glycoprotein lectin, resulted in destruction of up to 5 out of the 9 mannose residues present in each of its subunits (MW 30,000) without any loss of hemagglutinating activity. The oxidation did, however, abolish the interaction of soybean agglutinin with concanvalin A, as measured by quantitative precipitation. Reduction with sodium [3-H]borohydride of soybean agglutinin in which 4 out of 9 mannose residues per subunit were oxidized, afforded a radioactive product which retained full hemagglutinating activity and was indistinguishable from the native lectin by gel filtration, gel electrophoresis, and affinity chromatography. These results establish that the integrity of the carbohydrate side chain of soybean agglutinin is not essential for the biological activity of the lectin, and suggest a general method for the preparation of radioactive glycoprotein lectins.     

Sites of action of D2O in intact and skinned crayfish muscle fibers

Summary The effect on tension development of replacing 90% of the H₂O of the bathing saline with D₂O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D₂O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1–2 orders of magnitude smaller in D₂O than in H₂O saline. When bathed in D₂O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D₂O salines.     

Studies on the mechanism of action of hexamethylene bisacetamide, a potent inducer of erythroleukemic differentiation.

Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up by the cell; an intracellular concentration equal to the extracellular concentration is achieved by 6-8 h. Commitment to differentiation is not detected until at least 10 h after equilibration. Both uptake and commitment to differentiate are concentration and temperature dependent. The majority of the compound is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in both cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, but acetylation of the diamine greatly increases inducing activity.     

Proteomic profiling and functional characterization of early and late shoulder osteoarthritis

Introduction

The development of effective treatments for osteoarthritis (OA) has been hampered by a poor understanding of OA at the cellular and molecular levels. Emerging as a disease of the ''whole joint’, the importance of the biochemical contribution of various tissues, including synovium, bone and articular cartilage, has become increasingly significant. Bathing the entire joint structure, the proteomic analysis of synovial fluid (SF) from osteoarthritic shoulders offers a valuable ''snapshot’ of the biologic environment throughout disease progression. The purpose of this study was to identify differentially expressed proteins in early and late shoulder osteoarthritic SF in comparison to healthy SF.

Methods

A quantitative ¹⁸O labeling proteomic approach was employed to identify the dysregulated SF proteins in early (n = 5) and late (n = 4) OA patients compared to control individuals (n = 5). In addition, ELISA was used to quantify six pro-inflammatory and two anti-inflammatory cytokines.

Results

Key results include a greater relative abundance of proteins related to the complement system and the extracellular matrix in SF from both early and late OA. Pathway analyses suggests dysregulation of the acute phase response, liver x receptor/retinoid x receptor (LXR/RXR), complement system and coagulation pathways in both early and late OA. The network related to lipid metabolism was down-regulated in both early and late OA. Inflammatory cytokines including interleukin (IL) 6, IL 8 and IL 18 were up-regulated in early and late OA.

Conclusions

The results suggest a dysregulation of wound repair pathways in shoulder OA contributing to the presence of a ''chronic wound’ that progresses irreversibly from early to later stages of OA. Protease inhibitors were downregulated in late OA suggesting uncontrolled proteolytic activity occurring in late OA. These results contribute to the theory that protease inhibitors represent promising therapeutic agents which could limit proteolytic activity that ultimately leads to cartilage destruction.     

Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme’s capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.     

Molecular Detection of Ehrlichia canis in Dogs in Malaysia

An epidemiological study of Ehrlichia canis infection in dogs in Peninsular Malaysia was carried out using molecular detection techniques. A total of 500 canine blood samples were collected from veterinary clinics and dog shelters. Molecular screening by polymerase chain reaction (PCR) was performed using genus-specific primers followed by PCR using E. canis species-specific primers. Ten out of 500 dogs were positive for E. canis. A phylogenetic analysis of the E. canis Malaysia strain showed that it was grouped tightly with other E. canis strains from different geographic regions. The present study revealed for the first time, the presence of genetically confirmed E. canis with a prevalence rate of 2.0% in naturally infected dogs in Malaysia.     

Impact of H216 on the DNA Binding and Catalytic Activities of the HIV Restriction Factor APOBEC3G

APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd''s deamination reactions within a 5′-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5′-CCC substrate over 5′-CCU substrate, which explains why A3G displays almost no processivity within a 5′-CCC motif. In addition, A3Gctd''s shortest substrate sequence was found to be a pentanucleotide containing 5′-CCC flanked on both sides by a single nucleotide. A3Gctd as well as full-length A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo.