Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

Strain differences to effects of aging on concentrations of amino acids in cerebrospinal fluid between Sprague Dawley rat and Wistar Kyoto rat.

Age-related alterations and differences of weights and those of amino acid concentrations in the cerebrospinal fluid (CSF) were evaluated between Sprague Dawley (SD) rats and Wistar Kyoto (WKY) rats from eight to twenty weeks of age. The weights of SD rats were heavier than WKY rats at all ages. The age-related alterations of the CSF concentration of many amino acids within each strain were significant but showed no significant trend with age. Between the strains, the concentration differences of excitatory and inhibitory amino acids were not frequent although the concentrations of arginine, alanine and threonine were significantly higher in SD rats than in WKY rats. These results suggest that the different CSF concentrations of amino acids may relate to characteristics of rat strains.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Parasitic specialization ofGeotrichum candidum citrus race

Parasitic specialization ofGeotrichum candidum citrus race, the causal agent of citrus sour rot, was investigated. Of seven isolates tested for pathogenecity, all could infect ten species of citrus fruits and edible parts of five species of noncitrus crops. Only one isolate (Ap2), isolated from soil of an apple orchard, could infect apple fruit.     

Carcinogenesis in accessory sex organs of rats induced by 3,2′-dimethyl-4-aminobiphenyl: response to androgen deprivation

 It is generally accepted that early human prostate cancers reveal higher androgen dependency than do advanced ones. In the present study, we examined whether the animal model of prostate cancer has already lost androgen dependency at the early stages of carcinogenesis. At experimental week 46, androgen deprivation was induced in rats and the incidences of atypical hyperplasia and cancer were examined in the ventral, dorsolateral prostate, coagulating glands, and seminal vesicles. Androgen deprivation significantly lowered the incidence of atypical hyperplasia in all four organs. As for the incidence of cancer, no significant differences were observed in the coagulating glands and seminal vesicles. Regarding atypical hyperplasia, androgen deprivation significantly decreased the proliferative cell nuclear antigen labeling index in the coagulating gland and seminal vesicles. The presence of cancer was also decreased in the coagulating gland but not in the seminal vesicles. With control group specimens, more intense staining of androgen receptor was observed in atypical hyperplasias than in cancers. Compared with the atypical hyperplasias, the cancers revealed low androgen dependency at the early stages of carcinogenesis. The cancers in the seminal vesicles also revealed higher androgen independency than did those in the coagulating gland. Accepted: 6 May 1997     

Separation of Bombyxin from a neuropeptide of Bombyx mori showing Summer-morph-producing Hormone (SMPH) activity in the Asian Comma Butterfly,Polygonia c-aureum L

A neuropeptide from brain-suboesophageal ganglion (Br-SG) complexes of the silkmoth, Bombyx mori, shows summer-morph-producing hormone (SMPH) activity in the Asian comma butterfly, P. c-aureum. The SMPH-active peptide was extracted and demonstrated to be almost the same molecular size as bombyxin (4-5kD), a nueropeptide which shows prothoracicotropic hormone (PTTH) activity when assayed in vitro with prothoracic glands (PGs) of 4th-instar B. mori larvae in vitro. A Sephadex G-50 fraction of 3-8kD molecules prepared from Br-SG complexes of B. mori adults was applied to CM-, SP-, DEAE- or QAE- Toyoperal columns at pH 5.6 (or pH 6.9). The SMPH-activity could be separated from the PTTH-activity (or bombyxin) by subjecting a SMPH- and PTTH-active preparation of B. mori to anion-exchange chromatography at pH 6.9. By reversed-phase HPLC following an anion-exchange chromatography, SMPH-activity was recovered in two fractions of 40-45% acetonitril. Results demonstrate that the B. mori peptide showing the SMPH-activity in P. c-aureum is a different molecule than bombyxin.