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Excess cholesterol is associated with cardiovascular diseases (CVD), an important cause of mortality worldwide. Current CVD therapeutic measures, lifestyle and dietary interventions, and pharmaceutical agents for regulating cholesterol levels are inadequate. Probiotic bacteria have demonstrated potential to lower cholesterol levels by different mechanisms, including bile salt hydrolase activity, production of compounds that inhibit enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A, and cholesterol assimilation. This work investigates 11 Lactobacillus strains for cholesterol assimilation. Probiotic strains for investigation were selected from the literature: Lactobacillus reuteri NCIMB 11951, L. reuteri NCIMB 701359, L. reuteri NCIMB 702655, L. reuteri NCIMB 701089, L. reuteri NCIMB 702656, Lactobacillus fermentum NCIMB 5221, L. fermentum NCIMB 8829, L. fermentum NCIMB 2797, Lactobacillus rhamnosus ATCC 53103 GG, Lactobacillus acidophilus ATCC 314, and Lactobacillus plantarum ATCC 14917. Cholesterol assimilation was investigated in culture media and under simulated intestinal conditions. The best cholesterol assimilator was L. plantarum ATCC 14917 (15.18 ± 0.55 mg/1010 cfu) in MRS broth. L. reuteri NCIMB 701089 assimilated over 67% (2254.70 ± 63.33 mg/1010 cfu) of cholesterol, the most of all the strains, under intestinal conditions. This work demonstrates that probiotic bacteria can assimilate cholesterol under intestinal conditions, with L. reuteri NCIMB 701089 showing great potential as a CVD therapeutic.

Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme [E2(14)k; Wing, Dumas and Banville (1992) J. Biol. Chem. 267, 6495-6501] that efficiently supported ubiquitination and protein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins. This suggested that mammalian homologues of UBC4/UBC5 remained to be identified. Using oligonucleotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16,700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k. Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis. Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins. One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism. This paper reports the first cloning of a apparent mammalian homologue of S. cerevisiae UBC4/UBC5. Its high expression in testis and ability to efficiently support conjugation to testis proteins suggest that this family of E2s may play a role in the proteolysis that occurs during spermatogenesis.

In this study, a serotonin−stearic acid (ST-SA)-based bioconjugate was synthesized for the surface modification of manganese oxide-based nanocuboids (MNCs) for delivering of anticancer drug (i.e., doxorubicin hydrochloride (DOX)) to human liver cancer cells. MNCs were synthesized by chemical precipitation method, and their surface was modified with ST-SA bioconjugate for targeting of MNCs to cancer cells. The ST-SA@MNCs along with DOX showed good colloidal stability, high drug encapsulation (98.3%), and drug loading efficiencies (22.9%) as well as pH-responsive biodegradation. Coating with ST-SA conjugate provided a shield to MNCs which sustained their degradation in an acidic environment. The release of DOX was higher (81.4%) in acidic media than under the physiological conditions (20.5%) up to 192 h. The in vitro antiproliferation assay showed that ST-SA@MNCs exhibit higher cell growth inhibition compared to that of pure DOX after 48 h of treatment. The cellular uptake and apoptosis studies revealed the enhanced uptake of ST-SA@MNCs in contrast to the MNCs due to overexpressed ST receptor on hepatocellular carcinoma cells and triggered the generation of reactive oxygen species in the cells. Therefore, these results indicated that the DOX-loaded, ST-SA stabilized MNCs improved the therapeutic index of DOX and would be a promising therapeutic candidate for tumor therapy.

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5-Fluorouracil (5-FU) is one of the most prescribed drugs and the major component of chemotherapy for the treatment of colorectal cancer. In this study, we have designed arginine-functionalized manganese oxide nanocuboids (Arg@MNCs) for the effective delivery of 5-FU to colon cancer cells. Arginine was used as multifunctional agent to provide stability to MNCs, achieve high drug loading, control the release of loaded drug, and improve delivery to cancer cells. The synthesized Arg@MNCs were characterized by DLS, TEM, XRD, FTIR, XPS, TGA, and VSM analysis. The structural and morphological analysis by TEM showed cuboid-shaped MNCs with average particle size ∼15 nm. Biodegradation studies indicated that the Arg@MNCs were degraded at endolyosomal pH in 24 h while remaining stable at physiological pH. Hemolytic toxicity studies revealed the safety and nontoxic nature of the prepared MNCs. 5-FU-loaded Arg@MNCs showed significant control over the release of 5-FU, decrease in the hemolytic toxicity of loaded 5-FU but higher in vitro anticancer activity against HCT 116 and SW480 human colon cancer cells. Importantly, both the bare MNCs and Arg@MNCs showed excellent T1 and T2MR relaxivity under 3.0 T MRI scanner. Thus, the nanostructures developed in this study, i.e., 5-FU-Arg@MNCs could overcome the issues of both MNCs (stability) and 5-FU (low drug loading and nonspecificity) and may be used as a multifunctional theranostic nanocarrier for colon cancer treatment.