Video microscopy of colloidal gold particles and immuno-gold labelled microtubules in improved rectified DIC and epi-illumination

Modification of rectified Nomarski differential interference contrast optics (Nikon) and the epi-illumination system (Nikon IGS-cube) improved the detection of colloidal gold particles with analog video enhanced microscopy. Immuno-gold labelled microtubules of Haemanthus endosperm are visualized at a level of detection unmatched in conventional light microscopy. Single gold, or gold silver enhanced particles in suspension viewed with the modified epi-illumination after pressure injection into cells, are well distinguished from other granular cell components. Immuno-gold has also been detected on the surface of chromosomes and the nuclear envelopes in cells during the rapid experimental disassembly of microtubules. Thus, under certain conditions tubulin in a form other than microtubules may be detected. Practical applications of this "optical stain" for non fading immuno-gold 5-40 nm markers are discussed.     

Characterization of alpha-actinin from Acanthamoeba

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.     

Population of K-lymphocytes in various kinds of thyroid disease

We have measured the population of Killer (K) lymphocytes in the peripheral blood of 108 patients with various kinds of thyroid disease. In the patients with Hashimoto's thyroiditis and Graves' disease, the relative and absolute numbers of K-lymphocytes were significantly lower than those seen in healthy controls (p less than 0.001), and the longer the duration of medication, the lower the K-lymphocyte population. However, there was no apparent correlation between the serum titers of thyroid autoantibodies and the K-lymphocyte population. In the patients with malignant and benign thyroid tumors, the relative and absolute numbers of K-lymphocytes significantly decreased when compared with those of controls (p less than 0.001), the decrease was more prominent after surgical operation than before operation. A prominent decrease in the K-lymphocyte population was evoked to maximum 2 to 4 weeks after surgical operation. The patients with both malignant and benign tumors having abundant lymphocytic infiltration in their surgical specimens generally revealed a lower K-lymphocyte population than those having no lymphocytic infiltration.     

Peripheral resistance and red cell LiNa countertransport in borderline hypertensives

We have studied ouabain-resistant, external sodium-stimulated, lithium efflux (LiNa countertransport) in red blood cells from 21 borderline hypertensives with at least one hypertensive first degree relative (BH-F), 19 borderline hypertensives without family history of essential hypertension (BH-NF), and 35 age-matched normotensive subjects. The data indicate the finding of an increased LiNa countertransport in all BH (F+NF), but with a significant overlap between BH values and control ones: LiNa countertransport is significantly higher only in BH-F but it is normal in BH-NF. Moreover, there is a significant correlation of LiNa countertransport to total peripheral resistance but not to mean blood pressure in all hypertensive patients. It is suggested that in BH the increase of erythrocyte Na flux is mediated by the NaNa exchange diffusion, and its abnormality may be associated to the hereditary trait of essential hypertension rather than the high blood pressure per se, probably resulting in the development of hypertension, through the increased vascular smooth muscle tone.     

Chromosomal hypersensitivity in mutant M10 and Q31 mouse cells exposed to ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO)

2 mutant mouse cells M10 and Q31 were examined for chromosomal aberrations induced by ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO), as compared with mouse lymphoma L5178Y cells. Q31 cells are UV- and 4NQO-sensitive cells isolated from L5178Y cells. M10 cells are similar but are sensitive to ionizing radiation and 4NQO. After treatment with UV or 4NQO, chromatid-type aberrations in these cell strains were induced more frequently in the first mitotic cells, at late fixation times. After UV exposure (2.4 J/m2), the maximal frequencies of chromatid-type breaks in Q31 cells were about 5 times higher than in L5178Y cells. In M10 cells such breaks were only as frequent as in L5178Y cells. After 4NQO treatment (50 ng/ml) the frequencies of chromatid-type breaks in M10 and Q31 cells were significantly higher than in L5178Y cells. From these results and those of previous studies (Takahashi et al., 1982), M10 cells may be considered hypersensitive to gamma-rays and 4NQO, but not to UV, and thus react similarly to L5178Y cells. The hypersensitivity of M10 cells to 4NQO may result from a defect in the ionizing-radiation repair mechanism as has been suggested to occur in ataxia telangiectasia (AT) cells. Q31 cells are hypersensitive to UV and 4NQO, but not to gamma-rays. Q31 cells may be considered to be deficient in a UV-like repair pathway. In conclusion, characteristics of murine M10 and Q31 cells are compared with those of human AT and xeroderma pigmentosum (XP) cells.     

Hydropiperoside,a novel coumaryl glycoside from the root of Polygonum hydropiper

From the methanol extract of the root of Polygonum hydropiper, a novel coumaryl glycoside hydropiperoside was isolated together with anthraquinone, ellagic acid 3,3′-di-O-methyl ether, gallic acid, two quercetin glycosides and an unidentified aromatic δ-lactone possessing antifertility activity. The structure of hydropiperoside was established as β-d-(1,3,6-tri-p-coumaryl)-fructofuranosyl-α-d-glucopyranoside by combination of extensive ¹H NMR and ¹³C NMR spectra, and the FD/MS spectrum.     

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.