Cultured circular smooth muscle from the rabbit colon

Summary Although cultured vascular smooth muscle cells have been extensively characterized and investigated, there are very few studies of cultured intestinal smooth muscle cells. The aim of this study was to culture colonic smooth muscle (CSM) cells from the rabbit colon. Freshly isolated CSM cells from the circular muscle layer of the distal colon were prepared by collagenase digestion. In primary culture, CSM cells attached to the culture vessels by 48 to 72 h, proliferated by 3 to 7 d, and reached confluency by 14 to 17 d with a “hill-and-valley” pattern. Spontaneous contractions were not observed at any time at 21° or 37° C. Confluent primary cultures were greater than 95% CSM cells, as identified by intensely positive immunofluorescent staining to smooth muscle actin-specific CGA7 and muscle-specific HHF-35 monoclonal antibodies. Transmission electron microscopy of freshly isolated and proliferating CSM cells revealed ultrastructural features consistent with smooth muscle cells. We successfully cultured CSM cells of the rabbit from freshly isolated cells and validated these CSM cells by electron microscopy and immunocytochemical staining. These highly pure primary cultures may be used to investigate numerous aspects of CSM cell metabolism and physiology. These studies were supported by the National Institutes of Health grant to the Inflammatory Bowel Disease Center (Bethesda, MD) P30-AM-32200 and R01-DK-31147. Dr. Kao is the recipient of a Research Career Development Award from the National Foundation for Ileitis and Colitis, Inc. A preliminary report of this work was presented at the American Motility Society Meeting, Houston, TX, in October 1986, and appeared in abstract form inGastroenterology 91: 1057; 1986.     

Similar C-phycocyanins from two strains of thermotolerant cyanophyte Mastigocladus laminousus.

C-phycocyanin from two strains of the thermotolerant blue-green alga, Mastigocladus laminosus (NZ-DB2-m and I-30-m), that grow within different temperature ranges have been characterized with respect to aggregation, immunologic properties, subunit composition, and thermodenaturation. The critical thermal-denaturation temperature for phycocyanin from both strains of M. laminosus phycocyanin is 60 degrees C which is higher than that for mesophilic phyococyanin. Immunodiffusion studied have shown that these two strains of M. laminosus exhibit no antigenic differences and are closely related to the mesophilic Plectonema calothricoides and the thermophilic Synechococcus lividus (strains 3). Neither phenol nor alpha-naphthol has any effect on phycocyanin aggregation in these two strains of M. laminosus. There is also no enhancement of formation of large aggregates at their elevated temperature of cultivation. Furthermore, the phycocyanin of both strains of M. laminosus does not demonstrate any large amount of 19S or higher aggregates at any pH value. These observations suggest that the mode of adaptation of M. laminosus phycocyanin to high temperature is differnet from the previously encountered. It is also important to note that phycocyanin is essentially unchanged whether it is extracted from the same strain, M. laminosus (NZ-DBS-m), grown at either 50 degrees C or 37 degrees C.     

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Pre‐copulation intervals,copulation frequencies,and initial progeny sex ratios in two biotypes of whitefly,Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) is a species complex, and its systematic classification requires controlled crossing experiments among its genetic groups. Accurate information on pre‐copulation intervals, copulation frequencies, and initial frequency of egg fertilization of newly emerged adults is critical for designing procedures for collecting the virgin adults necessary for these experiments. In the literature, considerable variation is reported between B. tabaci populations, with respect to the length of the pre‐copulation interval and the initial frequency of egg fertilization. Here, we used a video‐recording method to observe continuously the copulation behaviour of the Mediterranean/Asia Minor/Africa (B biotype) and the Asia II (ZHJ1 biotype) groups of B. tabaci. We also recorded the initial frequency of egg fertilization, as determined by the sex of the progeny. When adults were caged in female–male pairs on leaves of cotton plants, the earliest copulation events occurred 2–6 h after emergence; at 12 h after emergence 56–84% of the females had copulated at least once, and nearly all (92–100%) had copulated at least once by 36 h after emergence. Both females and males copulated repeatedly. Approximately 80 and 20% of copulation events occurred during the photophase and scotophase, respectively. By 72 h post‐emergence, the females of the B and ZHJ1 biotypes had copulated on average 6.1 and 3.9 times, respectively. When adults were caged in groups on plants 1–13 h after emergence, 30–35% of the eggs deposited during this period were fertilized, and approximately 90% of females were fertilized by the end of the 13 h. Although timing of copulation differed in detail between the two genetic groups, the results demonstrate that B. tabaci adults can start to copulate as early as 2–6 h post‐emergence and the majority of females can become fertilized on the day that they emerge.     

Lipopeptides of the N-terminus of Escherichia coli lipoprotein: synthesis, mitogenicity and properties in monolayer experiments

The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-L-Cys-Ser and analogs with longer sequences, are polyclonal activators for B-lymphocytes. Triple-chain lipopeptides also constitute efficient low-molecular-weight carrier/adjuvant systems, which can be linked to antigens to yield immunogens for antibody production without further additives. This is the first report of monolayer experiments with chemically well defined, synthetic lipopeptide mitogens with the composition of the N-terminus of an important bacterial membrane protein. Various derivatives of the lipoprotein N-terminus were synthesized. These lipopeptides differed in the length of the peptide moiety, the number of fatty acid residues, and protective groups. In order to obtain the surface areas for the lipopeptides in isotherms and hysteresis isotherms, monolayer experiments with a computer-controlled film balance were performed. To get some information about the interaction of these compounds with typical membrane lipids mixed monolayers were formed from triple-chain lipopeptides with dipalmitoylphosphatidylcholine and cholesterol. A comparison of the mitogenic response of the compounds was made in an in vitro system with B-lymphocytes from Balb/c mice.     

Electron Microscopic Studies of Spruce Needles in Connection with the Occurrence of Novel Forest Decline

Needles of four spruce trees showing different degrees of novel kinds of forest decline were investigated by electron microscopy. Green needles appearing at least superficially still intact were selected for the present investigation. Most of the mesophyll appeared to be undamaged. However, groups of atypical mesophyll cells were found close to the endodermis or the hypodermis. The chloroplasts of the apparently damaged cells were particularly affected. Changes in the matrix of the chloroplasts, i.e,. increased affinity to osmium, occurrence of extensive nests of plastoglobuli, as well as damage to the membranes, i.e. lesions in the envelope and abnormal thylakoid membranes, were observed. Signs of decomposition of other cellular structures including mitochondria were also detectable. There appeared to be a close correlation between the degree of damage at the whole tree level and the degree of damage occurring at the cellular level. It is concluded that particularly the lipids and the proteinsof, the membranes are affected by anthropogenic air pollutants and natural stressors. The altered membrane structure may for instance cause abnormal osmotic conditions for the cellular compartments and may impair transport processes and thus lead to lossof function not only of the cells but also of the whole needle.     

Isolation and chromosomal localization of the human En-2 gene

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.