Sequence analysis of porothramycin biosynthetic gene cluster

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.     

Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.     

The 14-3-3 protein affects the conformation of the regulatory domain of human tyrosine hydroxylase

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.     

Evaluation of natural antioxidants of Leuzea carthamoides as a result of a screening study of 88 plant extracts from the European Asteraceae and Cichoriaceae

In recently, there has been a great interest in natural antioxidants as bioactive components of food, nutraceuticals or potential drugs against several diseases. In our study, 88 extracts from various parts of plants from European Asteraceae and Cichoriaceae were assayed for radical scavenging activity by means of DPPH (1,1-diphenyl-2-picryl hydrazyl radical) test using the SIA (Sequential injection analysis) method developed for this purpose in our laboratory. DPPH radical scavenging activity of all tested plant extracts was evaluated according to the IC(50) parameter. 29 extracts exhibited IC(50) value lower than 0.1 mg/mL. The leaves of Leuzea carthamoides (IC(50) = 0.046 mg/mL) were chosen as the most promising sample for a subsequent phytochemical study, which resulted in isolation of seven natural compounds, namely, 4',5,7-trihydroxy-6-methoxyflavone (hispidulin) (1), 5, 7, 3', 4'- tetrahydroxyflavanone (eriodictyol) (2), 3',4',5,7-pentahydroxy-6-methoxyflavonol (patuletin) (3), eriodictyol-7-beta-glucopyranoside (4), 6-hydroxykaempferol-7-O-(6'-O-acetyl-beta-D-glucopyranoside) (5), 4-hydroxybenzoic acid (6) and 3,4-dihydroxybenzoic acid (protocatechuic acid) (7). Antioxidant activity of the isolated compounds was evaluated by DPPH test and ferric reducing antioxidant power (FRAP) test and compared with trolox and quercetin. Both tests evaluated the flavonoid (5) as the most active antioxidant. This result was confirmed by comparison with known data concerning the structure/activity relationships of flavonoids.     

Natural hybridization in tropical spikerushes of Eleocharis subgenus Limnochloa (Cyperaceae): Evidence from morphology and DNA markers

? Premise of the study: Natural hybridization represents an important force driving plant evolution and affecting community structure and functioning. Hybridization may be overlooked, however, among morphologically highly uniform congeners. An excellent example of such a group is Eleocharis subgenus Limnochloa, which has no reliably proven hybrids. Does this reflect biological barriers to interspecific crosses or difficulties in detecting the hybrids? We tested the hypothesis that hybridization occurs among sympatric Eleocharis cellulosa, E. interstincta, and E. mutata in northern Belize, Central America. ? Methods: Morphometric study (407 plants) was followed by examination of inter-simple sequence repeat (ISSR) polymorphisms (44 plants) and ITS sequence variation (33 plants). ? Key results: Two putatively hybrid morphotypes were discerned-E. cellulosa-resembling and E. interstincta-resembling. DNA markers of E. cellulosa and E. interstincta displayed additive constitution in plants from one E. cellulosa-resembling population only. The other putatively hybrid populations contained ISSR and ITS markers of the species they resembled morphologically, several unique ISSR markers, and ITS sequences of an undescribed South American Limnochloa entity. DNA markers of E. mutata were absent in the putative hybrids. ? Conclusions: Simultaneous use of various types of molecular markers can overcome many pitfalls of investigations concerning hybridization among closely related and morphologically similar species. Northern Belize represents a hybrid zone of E. cellulosa and E. interstincta. A third participant in the hybridization events occurring in this zone is an unknown Limnochloa lineage but is not E. mutata. Interspecific hybridization may play a significant role in the diversification of Eleocharis.     

Association between Low Levels of Mannan-Binding Lectin and Markers of Autoimmune Thyroid Disease in Pregnancy

Functional deficiency of mannan-binding lectin (MBL) has been associated with adverse pregnancy outcome. Adverse events during pregnancy have also been described in women with autoimmune thyroid diseases (AITD), and thyroid hormones have been shown to influence serum levels of MBL. Therefore, the aim of this study was to analyse the impact of MBL-deficiency on the outcome of pregnancy in relation to the presence of AITD. Almost one year after delivery, we assessed serum MBL levels and MBL2-genotypes in 212 women positively screened for AITD in pregnancy. In 103 of these women, we could also measure MBL levels in frozen serum samples from the 9-12^th gestational week, obtaining 96 pairs of MBL values (pregnancy vs. follow-up). As controls, 80 sera of pregnant women screened negatively for AITD were used. MBL2-genotyping was performed using multiplex PCR. Women with thyroid dysfunction and/or thyroid peroxidase antibodies (TPOAb) had lower MBL levels during pregnancy than controls, (3275 vs. 5000 ng/ml, p<0.05). The lowest levels were found in women with elevated thyroid-stimulating hormone (TSH) levels in the absence of TPOAb (2207 ng/ml; p<0.01 as compared to controls). MBL2 genotype distribution did not differ between subgroups. At a median follow-up period of 17 months (range: 3–78 months) after delivery, median MBL level had decreased further to 1923 ng/ml (p<0.0001) without significant changes in TSH. In an explorative survey, functional MBL-deficiency was neither linked to a history of spontaneous abortion, nor other obstetric complications, severe infections throughout life/pregnancy or antibiotics use in pregnancy. In conclusion, hypothyroidism during pregnancy is associated with decreased MBL levels, and the levels decreased further after delivery.     

Mycobacterium avium Subspecies paratuberculosis Cultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Detection of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic

We investigated the presence of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure to M. avium subsp. paratuberculosis, albeit at a low level for viable cells.