Differential expression patterns of SUMO proteins in HL-60 cancer cell lines support a role for sumoylation in the development of drug resistance

Small ubiquitin-like modifier (SUMO) proteins are involved in a variety of cellular processes. Alterations in SUMO conjugation have been implicated in several human diseases, including cancer. Although the main cause of failure in cancer treatment is the development of drug resistance by cancer cells, the mechanisms of drug resistance are not fully understood. SUMO proteins are thought to play roles in various cellular pathways, but no studies have as yet compared the expression of the different SUMO proteins in chemosensitive and drug-resistant cancer cells. To determine the relationship between protein sumoylation and drug resistance, the expression of various SUMO isoforms has been studied and compared in the HL-60 cell line (a model for leukemic cells) and in HL-60RV cells (resistant to vincristine). Co-immunostaining of cells by anti-SUMO antibodies and antibodies against various nuclear subdomains has been examined by an advanced type of bioimaging analysis. Whereas SUMO-2/3 co-localizes exclusively with nuclear bodies containing promyelocytic leukemia protein in both cell types, SUMO-1 has also been seen in nucleolar regions of HL-60, but not in HL-60RV, cells. In HL-60 cells, SUMO-1 occurs adjacent to, but not co-localized with, the nucleolar marker fibrillarin. Western blot analysis has revealed higher levels of free SUMO and sumoylated products in drug-resistant cells and the presence of specific SUMO-1 conjugates in drug-sensitive HL-60 cells, possibly consistent with a specific nucleolar signal. Shortly after the induction of ethanol and oxidative stress, HL-60RV, but not HL-60, cells show increased accumulation of high-molecular-weight SUMO-2/3 conjugates. Thus, SUMO-1 probably has a specific role in the nucleoli of HL-60 cells, and the alteration of sumoylation might be a contributing factor in the development of drug resistance in leukemia cells. The author thanks Stern College for Women, Yeshiva University and the Joseph Alexander Foundation for supporting this research project.     

The use of previous crops as sustainable and eco-friendly management to fight Fusarium oxysporum in sesame plants

Sesame (Sesamum indicum L.), the “Queen of oil seeds” is being infected with pathogens, i.e., fungi, bacteria, virus and nematodes. Fusarium oxysporum sp. sesami (Zap.), is one of the fiercest pathogens causing severe economic losses on sesame. This work aimed to evaluate the impact of the cultivation of some preceding crops and seed inoculation with antagonistic predominant rhizospheric bacteria and actinomycetes on the incidence and development of Fusarium damping-off and wilt disease. Results showed that the lowest pre and/or post-emergence damping-off and wilt of sesame were recorded after onion and garlic, followed by wheat compared to clover in both the 2019 and 2020 seasons. In vitro, soil extracts from plots where onion and garlic have been cultivated slightly decreased the conidia germination and mycelium radial growth of F. oxysporum. The numbers of sesame rhizospheric F. oxysporum and fungi were lower after the cultivation of onion and garlic than those after wheat and clover. However, the numbers of actinomycetes and bacteria were higher in the onion, garlic, and clover rhizosphere than wheat. Among all isolated bacteria and actinomycetes associated with sesame roots cultivated after preceding plants, the Tricoderma viride and Bacillus subtilis (isolate No.3) profoundly reduce F. oxysporum mycelial growth in vitro. When sesame seeds were inoculated with Tricoderma viride, Bacillus subtilis, Streptomyces rochei and Pseudomonas fluorescens, the disease incidence of damping-off and wilt significantly decreased in the greenhouse and field trials conducted in both tested growing seasons, also had highly significant on plant health and growth parameters. Therefore, the current study suggested that using the preceding onion and garlic plants could be used for eco-friendly reduction of damping-off and wilt disease of sesame.     

Evaluation of Herpes Simplex Detection in Corneal Scrapings by Three Molecular Methods

Herpes simplex virus (HSV)-1 keratitis (HSK) is a sight-threatening ocular infection with worldwide occurrence. A prompt laboratory diagnosis is often very useful. The purpose of this study was to evaluate molecular methods as rapid diagnostic tools compared with cell culture of HSK. Corneal scrapings from patients with clinically suspected HSK were tested by direct immunofluorescence assay (IFA) for HSV-1 antigen and by polymerase chain reaction (PCR) for HSV-1 DNA, and an attempt for viral isolation was performed on Vero cell line culture. Positive samples by cell culture were 20.8%, whereas PCR was positive in 29.2%, and IFA was positive in 33.3%. IFA had better sensitivity (80%) and negative predictive value (81.8%) than PCR (70% and 76.9%, respectively); however, PCR had better specificity (71.4%) and positive predictive value (63.6%). This indicates that a combination of cell culture, IFA and PCR constitutes the best set of tools for diagnosis of clinically suspected cases of HSK. Documented infection can be further assessed by cell-culture technique or PCR depending laboratory availability.     

N-acetylglucosamine-6-O-sulfotransferase-1: production in the baculovirus system and its applications to the synthesis of a sulfated oligosaccharide and to the modification of oligosaccharides in fibrinogen

N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the C-6 position of non-reducing GlcNAc. Human GlcNAc6ST-1 was expressed as a fusion protein with protein A in an insect cell line (Tn 5 cells) using the baculovirus system. The recombinant enzyme was purified to homogeneity by IgG Sepharose column chromatography. The substrate specificity and the kinetic properties of the enzyme were similar to those of the enzyme expressed in the mammalian system. The purified recombinant enzyme was used to synthesize 6-sulfo GlcNAcbeta1-3Galbeta1-4Glc, which was identified by time of flight mass spectrometry. This sulfated trisaccharide served as a better substrate for microsomal galactosyltransferase from the mouse colon compared to 6-sulfo GlcNAc. The purified recombinant enzyme was also used to sulfate oligosaccharide chains on fibrinogen after enzymatic desialylation and degalactosylation to expose nonreducing GlcNAc residues. It is known that desialylation greatly increases the rate of clotting of fibrinogen after the addition of thrombin. Subsequent sulfation of desialylated and degalactosylated fibrinogen slightly decreased the rate of clotting. The recombinant GlcNAc6ST-1 is a useful reagent for 6-sulfate exposed GlcNAc residues both in oligosaccharides and in glycoproteins.     

Isolation and enumeration of marine actinomycetes from seawater and sediments in Alexandria

Actinomycetes were isolated from near-shore marine sediments and water at four different sites in Alexandria. Statistical analysis revealed that variation in temperature, pH, and dissolved phosphate were of insignificant values, but that variation in total nitrogen and organic matter were significant. The treatment of sediments and water samples by heat resulted in a selective reduction of the nonactinomycetal heterotrophic microflora. Four selective culture media were used for counting actinomycetes in marine water and sediments. The starch nitrate medium favored the growth of these microorganisms. The diversity and counts of actinomycetes varied with the seasonal variation, and the highest counts were detected in dry warm seasons. The numbers of this bacterial group in sediments exceeded by far their numbers in seawater. A positive correlation was found between population size and location. Actinomycetes were found in the highest numbers in the upper layers (0-20 cm depth). In a few cases, the counts of actinomycetes showed bimodal maxima 0-20 and 60-100 cm deep. Sediments were the best source of marine actinomycetes, and their distribution varied depending on the depth from which samples were collected. The ratio of actinomycetes to the total microflora ranged from 0.48 to 2.29, depending on location.     

Redox-silent tocotrienol esters as breast cancer proliferation and migration inhibitors

Tocotrienols are vitamin E members with potent antiproliferative activity against preneoplastic and neoplastic mammary epithelial cells with little or no effect on normal cell growth or functions. However, physicochemical and pharmacokinetic properties greatly limit their use as therapeutic agents. Tocotrienols' chemical instability, poor water solubility, NPC1L1-mediated transport, and rapid metabolism are examples of such obstacles which hinder the therapeutic use of these valuable natural products. Vitamin E esters like α-tocopheryl succinate were prepared to significantly improve chemical and metabolic stability, water solubility, and potency. Thus, 12 semisynthetic tocotrienol ester analogues 4-15 were prepared by direct esterification of natural tocotrienol isomers with various acid anhydrides or chlorides. Esters 4-15 were evaluated for their ability to inhibit the proliferation and migration of the mammary tumor cells +SA and MDA-MB-231, respectively. Esters 5, 9, and 11 effectively inhibited the proliferation of the highly metastatic +SA rodent mammary epithelial cells with IC(50) values of 0.62, 0.51, and 0.86μM, respectively, at doses that had no effect on immortalized normal mouse CL-S1 mammary epithelial cells. Esters 4, 6, 8-10, and 13 inhibited 50% of the migration of the human metastatic MDA-MB-231 breast cancer cells at a single 5μM dose in wound-healing assay. The most active ester 9 was 1000-fold more water-soluble and chemically stable versus its parent α-tocotrienol (1). These findings strongly suggest that redox-silent tocotrienol esters may provide superior therapeutic forms of tocotrienols for the control of metastatic breast cancer.     

Biochemical and biotechnological studies on a novel purified bacillus cholesterol oxidase tolerant to solvent and thermal stress

A novel bacterial strain was isolated and identified as Bacillus pumilus, with the capability to produce cholesterol oxidase enzyme (55?kDa). The production of the enzyme was optimized via two-step statistical approach. Out of eight factors screened in Plackett–Burman, only four had significant effects on enzyme activity. The optimization process of these four variables by Box–Behnken revealed that the maximum enzyme activity (90?U/mL) was significantly obtained after 6 days of fermentation with 0.3%, 1% and 0.2% of NH₄NO₃, yeast extract and Tween 80, respectively. The purified enzyme showed optimum activity at pH 7.5 and temperature of 40?°C. The enzyme retained 100% of its activity after storage at 40?°C for 60?min. The enzyme also exhibited enhanced stability in the presence of Tween 80, methanol and isopropanol. This solvent and thermal stress tolerant enzyme, produced by B. pumilus, may provide a practical option for industrial and analytical applications.     

Techniques to obtain uniformly growing young roots for the study of salt effects

Summary Two techniques are described for set ups to provide seedlings with regularly growing roots which may be used as biological test material for externally applied substances. The techniques were tried out with different plant species, and a model trial with wheat only is described.