Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.     

B-cell expansion in the presence of the novel 293-CD40L-sCD40L cell line allows the generation of large numbers of efficient xenoantigen-free APC

BACKGROUND: CD40-activated B lymphocytes have been used successfully as potent APC for the induction of T-cell responses. However, the 3T3-CD40L cell line, regularly used for engagement of CD40 on the B-cell surface, is a potential source of xenoantigens. This may affect the specificity of T cells stimulated with CD40-activated B cells, especially when generation of T-cell lines specific for endogenously processed Ag is desired. METHODS: To develop a system that allows efficient expansion of B cells in the absence of sources of xenoantigens, we created a human 293-CD40L-sCD40L cell line that produces soluble CD40L and expresses CD40L on the cell surface. B cells from patients with hematologic malignancies were expanded on the 293-CD40L-sCD40L cells and used for stimulation of either naive or in vivo primed donor T cells in three HLA-identical patient-donor combinations. RESULTS: The 293-CD40L-sCD40L cell line was able to stimulate B-cell growth with an efficiency superior to that of the commonly used 3T3-CD40L cell line. In all cases T-cell lines and, subsequently, T-cell clones were generated that showed reactivity against patient and not donor B cells, suggesting their specificity for minor histocompatibility antigens (mHAg). DISCUSSION: B cells activated with GMP grade 293-CD40L-sCD40L can be used in a variety of applications. In particular, they may be suitable for ex vivo stimulation of T cells prior to donor lymphocyte infusion (DLI), which may enhance its graft versus leukemia (GvL) effect.     

Sustained Protein Kinase D Activation Mediates Respiratory Syncytial Virus-Induced Airway Barrier Disruption

Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.     

The Absence of Non-Local Conformational Changes in OR3 Operator DNA on Complexing with the Cro Repressor

Abstract

The Interaction of the cro protein of λ phage with a synthetic OR3 operator having 17 base pairs in length and with its 9 bp fragment has been studied using the circular dichroism (CD) method. In both cases, a considerable change in the CD of the samples was found in the region 260-300 nm upon the addition of the cro protein. The stoichiometry obtained by the CD titration was identical for OR3 and its 9 bp fragment: one duplex per dimeric cro.

NaCl addition makes the complexes dissociate so that the 9 bp fragment becomes free at [NaCl]>0.2 M while the whole OR3 becomes free at [NaCl]>0.5 M.

The CD spectra of both the free duplexes show a typical B-form conservative pattern with a positive CD band (270 nm) and a negative one (250 nm). The specific complexing of both the duplexes results in a substantial CD depression in the positive band. The most pronounced effect occurs at 280 nm. This spectral change is quite distinct from those in the B to A transition and in the non-cooperative winding of the DNA within the B-family of forms.

The interaction of the cro protein with the non-operator DNAs, calf thymus DNA and a synthetic 10 bp duplex, reveals no visible CD changes at all.

An inference is drawn that the CD change in the specific complexes is mainly due to the induced CD in tyr-26 upon its interaction with a specific base pair in the operator or its fragment, the operator DNA conformation being conserved in a B-like form as a whole. However, some local distortions such as kinks cannot be ruled out on the basis of the CD data.     

Comparison of sensory structures on the antenna of different species of Philopotamidae (Insecta: Trichoptera)

Structure and distribution of sensilla were studied in sixteen species of the caddisfly family Philopotamidae. Their antennae bear numerous curved trichoid and pseudoplacoid sensilla and fewer coronal, styloconic and chaetoid sensilla on the flagellar segments. The most numerous pseudoplacoid sensilla have non-specific localization. The curved trichoid sensilla form clusters ventrally on each antennal segment. Sensilla belonging to coronal, styloconic and chaetoid types have specific positions. Long grooved trichoid sensilla are located nonspecifically in all the studied species. The average number of sensilla per segment decreases from the proximal to distal part of the flagellum. Scapus and pedicellum are devoid of most types of sensilla, however, they bear the Böhm bristles and long trichoid sensilla. A positive correlation between antenna dimensions and its cuticular structures is found.     

1-Benzyl derivatives of 5-(arylamino)uracils as anti-HIV-1 and anti-EBV agents

Pyrimidine analogs have long found use over a broad chemotherapeutic spectrum. In an effort to further explore the antiviral potential of several uracil derivatives previously synthesized in our laboratories, a series of benzylated pyrimidines were designed and synthesized. Introduction of the benzyl residue onto the 5-phenylaminouracil scaffold was carried out using 2,4-bis(trimethylsilyloxy)pyrimidine with the corresponding benzyl bromides. Similarly, 1-benzyl-5-(benzylamino)- and 1-benzyl-5-(phenethylamino)uracils were obtained via amination of 1-benzyl-5-bromouracils with benzylamine or phenylethylamine. The results of the broad screen antiviral studies revealed that compounds 5 and 11 exhibit promising inhibitory activity against HIV-1 in CEM-SS culture. A 50% protective effect was observed at concentrations of 11.9 and 9.5 μМ, respectively. Moreover, compounds 8 and 3 exhibited good inhibitory effects against EBV in АKАТА cell culture with EC₅₀ values of 2.3 and 12 μM, respectively. The synthesis and biological studies are detailed herein.     

Recurrent Emergence of Catalytically Inactive Ornithine Decarboxylase Homologous Forms That Likely Have Regulatory Function

Ornithine decarboxylase (ODC) catalyzes the first and rate limiting step in the biosynthesis of polyamines in most eukaryotes. Because polyamines have pleiotropic and often dramatic effects on cellular processes at both high and low concentrations, ODC expression is tightly controlled. ODC is regulated by a family of polyamine-induced proteins, antizymes, which bind to, and inactivate it. In mammals, and apparently most vertebrates, antizymes are in turn antagonized by proteins called antizyme inhibitors. Antizyme inhibitors are homologs of ODC that have lost their decarboxylation activity but have retained their ability to bind antizyme, in most cases even more tightly than ODC. We present a phylogenetic analysis of over 200 eukaryotic homologs of ODC and evaluate their potential to be either true ODCs or catalytically inactive proteins that might be analogs of the previously identified antizyme inhibitors. This analysis yielded several orthologous groups of putative novel antizyme inhibitors each apparently arising independently. In the process we also identify previously unrecognized ODC paralogs in several evolutionary branches, including a previously unrecognized ODC paralog in mammals, and we evaluate their biochemical potential based on their pattern of amino acid conservation.     

Marine mammals in macro-ecosystems of the Far Eastern seas and adjacent waters of the North Pacific

This article considers the role of marine mammals in a sea or ocean ecosystem based on the example of the Far Eastern seas with adjacent waters of the North Pacific, which is one of the regions of the World Ocean that is distinguished by its high biological and fish capacity and by a high abundance of cetaceans and pinnipeds. Based on extensive data, which was published mostly by Russian experts, the authors have estimated the quantities of annual consumption of fish and invertebrates by marine mammals in three Far Eastern seas: 14.6–18.2 million tons in the early 20th century; 12.3–15.1 million tons in the late 1970s; 22.7–28.8 million tons in the pre-harvest period; and 24.0–24.7 million tons in the early 21st century (27.0–29.5 million tons, if 3–5 million tons in ocean waters off the Kuril Islands and Kamchatka are taken into account). More than half of this quantity is formed by zooplankton and zoobenthos; the second largest portion consists of fish and squid. At the same time, the values of food consumption by fish and large invertebrates are much higher than these estimates for the 0–1000 m layer: 516 million tons were consumed in the 1980s–1990s; 389 million tons in 1991–1995; and 461 million tons in 1996–2005. In the years of high abundance, large walleye pollock alone consumed nearly 40 million tons of small fish and squid. Based on the data of 35-year-long ecosystem studies that were conducted by the Pacific Research Fisheries Center (TINRO Center), the following biomass estimates have been obtained for the biota of the Far Eastern Economic Zone of Russia: mesoand macroplankton, 1000 million tons; zoobenthos, 500 million tons; nekton, 100 million tons; benthic fish, 5 million tons; and large benthic invertebrates that are not included in the benthos, 2.43 million tons. By using these estimates and by comparing the quantities of food consumption by marine mammals, the conclusion was made that the role of marine mammals in food webs of waters of the Russian Far East is remarkable, but it does not reach a level that is high enough to regulate such a large-scale ecosystem as the macro-ecosystem of a sea or ocean.     

Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

Large‐scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale‐up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5‐KL production scale bioreactor compared to a 20‐L bench‐top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench‐top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5‐KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20‐L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.