Proceedings: Characteristics of freeze-dried cells

Microorganisms have been found to be more sensitive to selective media after freeze-drying. This increased sensitivity can be measured and thus the degree of sublethal injury can be determined as a function of various processing variables. In light of this, the use of selective media for the enrichment and detection of pathogens in freeze-dried products has to be reevaluated; indeed, the literature is now becoming abundant with such evaluations. In addition, the response of freeze-dried microorganisms has been found to be dependent on the medium in which they were grown; the phenomena of “metabolic injury” and “minimal medium recovery” are observed when microorganisms are grown in a complete and minimal medium, respectively. The expression of these two phenomena also can be used to assay for injury.Observations on the effects of freeze-drying on cell viability lead to the conclusion that freeze-drying is a complex stress. Damage to the cellular membrane structure and function, RNA integrity, and, possibly, DNA have been cited. The extrapolation of these macromolecular changes to specific viability responses for the purpose of elucidating the principal site of damage is still difficult. It is our opinion that the pre- and post-freeze-drying conditions to which the microorganisms are exposed can lead to a situation in which a particular macromolecular damage can become dominant over others, depending on the physiology of the cell.This knowledge can not only be applied for the purpose of improving detection of undesirable microbes but also for the preservation of desirable cultures, such as starter cultures in the dairy industry. Finally, the finding that microorganisms leak or release nucleic acids after freeze-drying, as they do after freezing and heating, can be applied to the problem of elimination of undesirable cytoplasmic components of organisms to be used as protein sources (4, 8).     

Improved methods for separating hydrolytic granules of neutrophil leucocytes

Highly efficient methods for isolating two hydrolytic granules of neutrophils are described. Neutrophil obtained from guinea pig peritoneal exudate cells were washed extensively with isotonic sucrose and then treated with heparin. More than 95 per cent of the cells so treated were disrupted with a Dounce homogenizer. Since nuclei were broken, leaving other organelles intact, homogenates were incubated with DNase to reduce viscosity. Postnuclear supernatants were centrifuged on a discontinuous gradient of Percoll. Azurophil granules, high in β-glucuronidase activity, sedimented at fractions of d = 1·081 and showed very little activity of other marker enzymes. High neutral α-glucosidase activity was observed in granular fractions of d = 1·038 and it is suggested that this is a marker for specific granules of neutrophils.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.     

Degradation of somatomedin A by various organ homogenates from rats

Degradative activities of somatomedin A (SMA) have been examined in various tissue homogenates of rat using trichloracetic acid precipitable radioactivity of 125I-SMA. Kidney and testis showed higher specific activities and liver and brain lower activities. They were dependent on SH reagents; 0.5 mM HgCl2 inhibited the degradative activity of liver completely and 1 mM dithiothreitol (DTT) augmented the activity slightly. The activities in liver were separated by differential centrifugation; about 90 per cent of the total activity in the whole homogenate was recovered in the supernatant fraction at 100,00 x g for 60 min, and 10 per cent in the precipitate. The pH profile of each fraction was different; that of the supernatant showed a single peak at pH 7.4 and that of the pellet revealed two peaks at pH 5.9 and 7.4. However, both fractions showed similar SH-dependency.     

Parafibromin expression in lung normal tissue and carcinoma: its comparison with clinicopathological parameters of carcinoma

Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its down-regulated expression is involved in the pathogenesis of parathyroid, gastric and colorectal carcinomas. To clarify the roles of parafibromin expression in lung carcinomas, it was examined by immunohistochemistry and in situ hybridization on tissue microarray containing lung carcinomas (n=144) and normal lung tissue (n=20), with a comparison to clinicopathological parameters of carcinomas. Lung carcinoma cell lines and tissues were studied for parafibromin expression by Western blot and RT-PCR. Down-regulated expression of parafibromin mRNA was found in lung carcinoma in comparison with matched normal tissue (p<0.05). Parafibromin protein was found in the cilia and nuclei of pseudo-stratified columnar epithelium, and the nuclei of lung carcinoma. According to immunostaining and in situ hybridization, there was no difference in parafibromin expression between histological subtypes of lung carcinoma (p>0.05). The Kaplan-Meier method indicated that nuclear parafibromin expression was positively correlated with adenocarcinoma patients (p<0.05). Down-regulated parafibromin mRNA expression might play an important role in lung carcinogenesis, but not in its histogenesis. Strong parafibromin expression in cilia of the pseudo-stratified columnar epithelium indicated its possible involvement in cell mobility. Parafibromin expression could be employed to indicate the favorable prognosis of patients with adenocarcinoma.     

Overexpression of GRP78 and GRP94 is involved in colorectal carcinogenesis

Glucose-related proteins (GRPs) are ubiquitously expressed in the endoplasmic reticulum and assist in protein folding and assembly, consequently considered to be molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in samples taken from several different malignant tissues. To clarify the roles of both molecules in tumorigenesis and progression of colorectal carcinomas, immunohistochemistry (IHC) was performed on tissue microarrays containing colorectal carcinomas, adenomas and the non-neoplastic mucosa (NNM) using antibodies against GRP78 and GRP94. Their expression was correlated with the clinicopathological parameters of carcinomas. Both proteins were also studied in colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) by IHC and Western blot. There was a gradually increased GRP78 expression from colorectal NNMs, carcinomas, to low-grade and high-grade adenomas (P<0.05), while up-regulated GRP94 expression from NNM, low-grade adenoma, high-grade adenoma, to carcinoma (P<0.05). The expression was similar in all the carcinoma cell lines. GRP78 expression was negatively correlated with lymphatic invasion or low GRP94 expression of the carcinomas (P<0.05), while there was no correlation of GRP94 expression with other parameters of carcinomas (P>0.05). Multivariate analysis showed that venous invasion, lymph node metastasis and UICC staging (P<0.05), but not age, sex, tumor size, differentiation, depth of invasion, lymphatic invasion, GRP78 and GRP94 expression (P>0.05), were independent prognostic factors for carcinomas. It is suggested that up-regulated expression of GRP78 and GRP94 could possibly be involved in the pathogenesis of colorectal carcinomas.     

Avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.     

Detection of KI polyomavirus and WU polyomavirus DNA by real-time polymerase chain reaction in nasopharyngeal swabs and in normal lung and lung adenocarcinoma tissues

Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.     

Identification of cytosine-phosphorothioate-guanine oligodeoxynucleotide sequences that induce interferon-γ production in feline immune cells

Unmethylated CpG-ODN are known to enhance Th1-type immune response. However, optimal sequences of CpG-ODN for activating Th1-type immune cells vary among species. It is necessary to identify the effective CpG-ODN sequences in each species. In the present study, in order to identify the sequences of CpG-ODN that produce fIFN-γ in cats, 14 kinds of ODN were synthesized and examined regarding their ability to induce fIFN-γ in feline PBMC and splenocytes. It was shown that some CpG-ODN significantly induced fIFN-γ production in splenocytes, but not in PBMC. We found that three kinds of CpG-ODN (no. 2, 5'-ggTGCATCGATGCAGggggG-3'; no. 5, 5'-ggTGCGTCGACGCAGggggG-3'; no. 10, 5'-ggTGCTACGTAGCAGggggG-3') specifically and significantly induced fIFN-γ production in feline splenocytes. The reverse sequences, GpC-ODN, do not cause significant fIFN-γ production. The fIFN-γ production inductivity of a mixture of CpG-ODN nos. 2, 5 and 10 was higher than those of individual CpG-ODN. When the CpG-ODN mixture was encapsulated in an MCL and administrated to cats, the number of fIFN-γ(+) cells in PBMC significantly increased. CpG-ODN nos. 2, 5 and 10 should be useful to elicit a Th1-type immune response as a vaccine adjuvant in cats.     

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.