A Cross-System Comparison of Bacterial and Fungal Biomass in Detritus Pools of Headwater Streams

The absolute amount of microbial biomass and relative contribution of fungi and bacteria are expected to vary among types of organic matter (OM) within a stream and will vary among streams because of differences in organic matter quality and quantity. Common types of benthic detritus [leaves, small wood, and fine benthic organic matter (FBOM)] were sampled in 9 small (1st-3rd order) streams selected to represent a range of important controlling factors such as surrounding vegetation, detritus standing stocks, and water chemistry. Direct counts of bacteria and measurements of ergosterol (a fungal sterol) were used to describe variation in bacterial and fungal biomass. There were significant differences in bacterial abundance among types of organic matter with higher densities per unit mass of organic matter on fine particles relative to either leaves or wood surfaces. In contrast, ergosterol concentrations were significantly greater on leaves and wood, confirming the predominance of fungal biomass in these larger size classes. In general, bacterial abundance per unit organic matter was less variable than fungal biomass, suggesting bacteria will be a more predictable component of stream microbial communities. For 7 of the 9 streams, the standing stock of fine benthic organic matter was large enough that habitat-weighted reach-scale bacterial biomass was equal to or greater than fungal biomass. The quantities of leaves and small wood varied among streams such that the relative contribution of reach-scale fungal biomass ranged from 10% to as much as 90% of microbial biomass. Ergosterol concentrations were positively associated with substrate C:N ratio while bacterial abundance was negatively correlated with C:N. Both these relationships are confounded by particle size, i.e., leaves and wood had higher C:N than fine benthic organic matter. There was a weak positive relationship between bacterial abundance and streamwater soluble reactive phosphorus concentration, but no apparent pattern between either bacteria or fungi and streamwater dissolved inorganic nitrogen. The variation in microbial biomass per unit organic matter and the relative abundance of different types of organic matter contributed equally to driving differences in total microbial biomass at the reach scale.     

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.     

Structural,conformational and thermodynamic aspects of groove-directed-intercalation of flavopiridol into DNA

Certain plant-derived alkaloids and flavonoids have shown propitious cytotoxic acitvity against different types of cancer, having deoxyribose nucleic acid (DNA) as their main cellular target. Flavopiridol, a semi-synthetic derivative of rohitukine (a natural compound isolated from Dysoxylum binectariferum plant), has attained much attention owing to its anticancer potential against various haematological malignancies and solid tumours. This work focuses on investigating interaction between flavopiridol and DNA at molecular level in order to decipher its underlying mechanism of action, which is not well understood. To define direct influence of flavopiridol on the structural, conformational and thermodynamic aspects of DNA, various spectroscopic and calorimetric techniques have been used. ATR-FTIR and SERS spectral outcomes indicate a novel insight into groove-directed-intercalation of flavopiridol into DNA via direct binding with nitrogenous bases guanine (C6=O6) and thymine (C2=O2) in DNA groove together with slight external binding to its sugar–phosphate backbone. Circular dichroism spectral analysis of flavopiridol–DNA complexes suggests perturbation in native B-conformation of DNA and its transition into C-form, which may be localized up to a few base pairs of DNA. UV–visible spectroscopic results illustrate dual binding mode of flavopiridol when interacts with DNA having association constant, K_a = 1.18 × 10⁴ M^?1. This suggests moderate type of interaction between flavopiridol and DNA. Further, UV melting analysis also supports spectroscopic outcomes. Thermodynamically, flavopiridol–DNA complexation is an enthalpy-driven exothermic process. These conclusions drawn from this study could be helpful in unveiling mechanism of cytoxicity induced by flavopiridol that can be further applied in the development of flavonoid-based new chemotherapeutics with more specificity and better efficacy.     

In vitro withanolide production by Withania somnifera L. cultures

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.     

Cellular Immune Responses to Cell Wall Peptidoglycan Associated Protein Antigens in Tuberculosis Patients and Healthy Subjects

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H₃₇Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD^?) and 11 PPD reactive healthy controls (HPPD⁺). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD⁺ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.     

Dermal gland secretion improves the heat tolerance of the brown dog tick, Rhipicephalus sanguineus, allowing for their prolonged exposure to host body temperature

Dermal glands (sensilla sagittiformia) secrete when brown dog ticks, Rhipicephalus sanguineus, are mechanically disturbed, presumably as a defensive mechanism. Recently, we observed that these glands secrete due to the pressure stimulation of engorgement. In this study, we examine how dermal gland secretion alters the physiology of R. sanguineus, particularly if this secretion is an important mechanism during blood feeding. The ability of ticks to retain water was not modified by dermal gland secretion, but heat tolerance was enhanced. Short-term heat shock was improved slightly (1 h at 50 °C to 1 h at 56 °C) and featured reduced injury responses and greater recovery after heat shock. When exposed to their host body temperature (37 °C) for prolonged periods, individuals that had secreted survived over 1 week longer than individuals that did not secrete. Dorsal application of squalene, the main component of dermal gland secretion, did not increase temperature tolerance, suggesting that the act of secreting rather than the physical properties of the secretion itself is responsible for the increase in heat tolerance. Based on our results, dermal gland secretion may be an essential mechanism in certain tick species (Amblyomma, Dermacentor, Hyalomma, Rhipicephalus, but not Ixodes) for tolerating body temperature and not succumbing to heat stress during the extended time periods of feeding on a mammalian host, serving as a mechanism to prevent heat damage from the host during feeding.     

Generation and identification of variants with improved purification yield of Bowman-Birk protease inhibitors carrying protein binding loops

Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduces the overall purification yield when BBI-AV is produced as a fusion protein in a Bacillus subtilis expression system. The low purification yield is primarily due to a higher fraction of molecules with incorrect disulfide bond configurations after production and also after disulfide bond shuffling induced by 2-mercaptoethanol. To improve production yields, site-saturation libraries were generated at 39 out of the 66 amino acid residues of BBI-AV. Initial screens were designed to select for variants with higher trypsin inhibitory activities than the parent after treatment with a reducing agent. Secondary screens were developed to select for variants with the highest purification yields, and to also eliminate any false positives. From the screens, it was found that positively charged substitutions in the exposed hydrophobic patch region (sites 27, 29, 40, 50 & 52) are especially productive. In fact, one substitution, F50R, improves the purification yield to nearly the same level as wild-type sBBI. Productive amino acid substitutions were combined to select for the variant with the best overall yield after purification. Several variants were obtained with higher purification yields than even sBBI. The octuple variants, A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T and A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q, are particularly productive having greater than a five fold increase in final purification yield over the parent.     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

Lead‐induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N‐acetyl cysteine and tannic acid

This study investigates the exposure of lead‐induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK‐2 cells). Following treatment of HK‐2 cells with an increasing concentration of lead nitrate (0–50 μM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose‐dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK‐2 cells. Lead exposure also induced apoptosis through caspase‐3 activation at 30 μg/mL. Pretreatment with N‐acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead‐induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase‐3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead‐induced DNA damage and apoptosis in an exposed person.     

Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

Summary An undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented with 10% fetal bovine serum (FBS). It is believed that certain, as yet unidentified, serum components play critical roles in the attachment and proliferation of MSCs. Commercially available FBS is poorly characterized and may vary in composition and quality from lot to lot. This study describes a method for the selection of lots of FBS that best support maintenance of the undifferentiated state, mitotic expansion of MSCsin vitro, and retention of multilineage developmental potential in response to appropriate cues.