Cellobiose dehydrogenase production by the mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus produces cellobiose dehydrogenase (CDHtc) in cellulose medium with the highest yield (55.88 U mL⁻¹) among all the reported fungal species. The enzyme has been isolated and purified from the culture filtrate.

CDHtc was found to be a very thermolabile enzyme with the temperature optimum at 30 °C, while it exhibited a wide range of pH stability from pH 2.0 to 8.0. Lactose was efficiently converted to lactobionic acid in presence of the enzyme. Addition of glucose in the cellulose medium on the first day of growth induced a lag period in enzyme production but ultimately facilitated earlier CDHtc production and the yield was also comparable to that achieved in the cellulose medium.     

Strong human endogenous retrovirus-specific T cell responses are associated with control of HIV-1 in chronic infection

Eight percent of the human genome is composed of human endogenous retroviruses (HERVs), which are thought to be inactive remnants of ancient infections. Previously, we showed that individuals with early HIV-1 infection have stronger anti-HERV T cell responses than uninfected controls. In this study, we investigated whether these responses persist in chronic HIV-1 infection and whether they have a role in the control of HIV-1. Peripheral blood mononuclear cells (PBMCs) from 88 subjects diagnosed with HIV-1 infection for at least 1 year (median duration of diagnosis, 13 years) were tested for responses against HERV peptides in gamma interferon (IFN-γ) enzyme immunospot (ELISPOT) assays. Individuals who control HIV-1 viremia without highly active antiretroviral therapy (HAART) had stronger and broader HERV-specific T cell responses than HAART-suppressed patients, virologic noncontrollers, immunologic progressors, and uninfected controls (P < 0.05 for each pairwise comparison). In addition, the magnitude of the anti-HERV T cell response was inversely correlated with HIV-1 viral load (r(2) = 0.197, P = 0.0002) and associated with higher CD4(+) T cell counts (r(2) = 0.072, P = 0.027) in untreated patients. Flow cytometric analyses of an HLA-B51-restricted CD8(+) HERV response in one HIV-1-infected individual revealed a less activated and more differentiated phenotype than that stimulated by a homologous HIV-1 peptide. HLA-B51 tetramer dual staining within this individual confirmed two different T cell populations corresponding to these HERV and HIV-1 epitopes, ruling out cross-reactivity. These findings suggest a possible role for anti-HERV immunity in the control of chronic HIV-1 infection and provide support for a larger effort to design an HIV-1 vaccine that targets conserved antigens such as HERV.     

A Simple One Pot Purification of Bacterial Amylase From Fermented Broth Based on Affinity Toward Starch-Functionalized Magnetic Nanoparticle

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet–visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.     

Sequence of human DNA polymerase beta mRNA obtained through cDNA cloning

A cDNA library from polyA+ RNA of a human teratocarcinoma cell line in phage lambda gt11 was screened with a fragment of the rat beta-polymerase cDNA, lambda pol beta-10, as probe. Five positive phage were identified and plaque purified. The cDNA of one positive clone selected for detailed study was 1257 bp. This insert was sequenced and found to contain the coding region for beta-polymerase, as well as 163 bp and 137 bp from the 5' and 3' untranslated regions, respectively. The primary structure of human beta-polymerase (318 amino acids, Mr = 36, 133) deduced from the cDNA was similar to rat beta-polymerase (95% matched residues). The greatest difference between the sequences of the human and rat cDNAs was in the 3' untranslated regions (64% matched base residues). These results provide necessary sequence information for study of the human beta-polymerase gene.     

Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.

The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.     

Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells

The regulation of gelsolin levels during differentiation of the murine embryonal carcinoma cell line, PC-13, was investigated using nucleic acid and immunological probes. A cDNA clone, Mu-319, which contained the entire coding sequence for the cytoplasmic form of murine gelsolin was isolated using a polyclonal antibody. Gelsolin was detected in several cell lines but was not detectable in three undifferentiated embryonal carcinoma cell lines. Levels of gelsolin mRNA increased 10-fold during the differentiation of the murine embryonal carcinoma cell line, PC-13. Differentiation of PC-13 was accompanied by changes in cell shape, from small indistinct cells to large flat cells. The accumulation of gelsolin mRNA in PC-13 cells began 12-24 h after addition of the differentiation-inducing agents. In comparison, 2-5A-dependent RNase activity showed a 40-fold increase beginning after 24 to 36 h and c-fos mRNA were shown to increase about 9-fold beginning 36 to 60 h after induction of differentiation. The levels of gelsolin per se, as determined by immunoreactivity were also shown to increase with differentiation of PC-13 cells. These results suggest that gelsolin may play a role in the restructuring of actin filaments which accompanies the dramatic changes in cell shape during differentiation.