Adaptation of mouse-human hybridomas to a protein free medium

Summary Five mouse-human hybridoma cell lines secreting human IgM class monoclonal antibodies were examined for possible adaptation to a protein free medium. Four were successfully adapted to the medium. The adapted cell lines could be cultured for more than two months without any changes in their cell growth abilities.     

Transport across the bacterial outer membrane

Diffusion of small molecules across the outer membrane of gram-negative bacteria may occur through protein channels and through lipid bilayer domains. Among protein channels, many examples of trimeric porins, which produce water-filled diffusion channels, are known. Although the channels are nonspecific, the diffusion rates of solutes are often drastically affected by their gross physicochemical properties, such as size, charge, or lipophilicity, because the channel has a dimension not too different from that of the diffusing solutes. In the last few years, the structures of three such porins have been solved by X-ray crystallography. It is now known that a monomer unit traverses the membrane 16 times as -strands, and one of the external loop folds back into the channel to produce a narrow constriction. Most of the static properties of the channel, such as the pore size and the position of the amino acids that produce the constriction, can now be explained by the three-dimensional structure. Controversy, however, still surrounds the issue of whether there are dynamic modulation of the channel properties in response to pH, ionic strength, or membrane potential, and of whether such responses are physiological. More recently, two examples of monomeric porins have been identified. These porins allow a very slow diffusion of solutes, but the reason for this low permeability is still unclear. Finally, channels with specific binding sites facilitate the diffusion of specific classes of nutrients, often those compounds that are too large to penetrate rapidly through the porin channels. Lipid bilayers in the outer membrane were shown to be perhaps 50- to 100-fold less permeable to uncharged, lipophilic molecules in comparison with the bilayers made of the usual glycerophospholipids. This is caused by the presence of a lipopolysaccharide leaflet in the bilayer, and more specifically, by the presence of a larger number of fatty acids in each lipid molecule, and by the absence of unsaturated fatty acids in the lipopolysaccharide structure.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide.

The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+-ATPase by cloned genes from the same and different species

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha-subunit gene of the F₁F₀ type of H⁺-ATPase was deleted. MS117 had low H⁺-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha-subunit gene of E. hirae H⁺-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F₁F₀-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H⁺-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F₁F₀ type of H⁺-ATPase.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.