C1q,a collagen-like complement subcomponent,in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.     

Induction of tumoricidal activated macrophages by a liposome-encapsulated glycolipid, trehalose 2,3,6'-trimycolate from Gordona aurantiaca

A mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate, prepared from a non-pathogenic acid-fast bacterium Gordona aurantiaca, was shown to induce strong tumoricidal activity in peritoneal exudate cells by intravenous or intraperitoneal injection of liposome-encapsulated preparations. The mycolic acid derivative containing a high proportion of unsaturated fatty acids rendered macrophages cytotoxic against syngeneic mastocytoma cells in the absence of endotoxin, for over 14 days after the injection. The macrophages were ascertained to be at low intracellular levels of a lysosomal enzyme beta-galactosidase and an ectoenzyme alkaline phosphodiesterase, a specific pattern as previously described for "primed macrophages". However the culture supernatants of the peritoneal exudate cells were not cytotoxic.     

Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum

Abstract A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of λgt10. The DNA insert of a hemolysin-positive (Hly⁺) λ-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly⁺ phenotype on E. coli . Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an α toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the α toxin secreted by C. septicum . Taken together, the data indicated that plasmid pCS21 indeed encoded an α toxin gene of C. septicum .     

In vitro synthesis of ornithine transcarbamylase.

An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.     

Comparative studies on antibody formation against hen egg white lysozyme and its split fragment.

A peptide fragment Fr. 17 (Lys1-Cys-Asn27 Leu129-Cys-Ala122) of hen egg white lysozyme (HL) was previously found to retain at least one antigenic determinant region of the native protein. In this work a highly purified preparation of Fr. 17, contaminated with less than 0.01% HL and less than 1% of other fragments was found to be strongly immunogenic to rabbits. The kinetic patterns of antibody formation against Fr.17, assayed by passive hemagglutination (PHA), were quite different from those of antibody formation against HL. The specificity of the antibody elicited to Fr. 17 was mainly directed to the Fr. 9-10-a region (Ala11-Asn27) while that of the antibody elicited to the Fr. 17 region in native HL was directed to the Fr. 15-b region (Lys1-Cys-Ala10 Leu129-Cys-Trp123). It is concluded that in the process of antibody formation, the recognition of the Fr. 17 region in native HL is different from that of fragment Fr. 17.     

Ribosome induces transdifferentiation of A549 and H-111-TC cancer cell lines

Previously we reported that, lactic acid bacteria (LAB) can induce human dermal fibroblast (HDF) cells to form multipotent cell clusters which are able to transdifferentiate into three germ layer derived cell lineages. Later on, we confirmed that ribosome is responsible for the LAB-induced transdifferentiation and ribosomes from diverse organisms can mimic the LAB effect on HDF cells. In our present study we have shown that, upon incorporation of ribosomes, non-small cell lung cancer cell line A549 and gastric tubular adenocarcinoma cell line H-111-TC are transformed into spheroid like morphology those can be transdifferentiated into adipocytes and osteoblast. Our qPCR analysis has revealed that, during the formation of ribosome induced cancer cell spheroids, the expression of the cancer cell associated markers and cell cycle/proliferation markers were altered at different time point. Through our investigation, here we report a novel and a non-invasive approach for cancer cell reprogramming by incorporating ribosomes.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.