Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli

Agarose gel electrophoresis of the plasmid deoxyribonucleic acids from 60 gram-negative bacilli recovered during investigations of nosocomial epidemics was used to fingerprint the strains. This method was as specific at differentiating bacterial strains as more conventional phenotyping methods. In all cases, plasmid band fingerprints of epidermic strains isolates were identical whereas coisolate plasmid deoxyribonucleic acid patterns were different. Agarose gel electrophoresis of plasmid deoxyribonucleic acid is proposed as a method which can be used in conventional microbiology laboratories as an adjunct to or, possibly, replacement for other methods of identifying bacterial strains.     

Cortisol,acth, and biosynthesis of apolipoproteins in rat hepatocytes

The effect of cortisol (5 mg/kg, 5 and 10 days) on biosynthesis of apoproteins of very low density lipoproteins in the liver and on synthesis of apoproteins of very low, low, and high density lipoproteins in blood serum of intact animals was investigatedin vivo. Cortisol, within the periods specified, inhibits biosynthesis of apoproteins of very low density lipoproteins (apo-VLDL) in liver. After adrenalectomy apo-VLDL synthesis is intensified and this effect is abolished during replacement administration, of cortisol. Apoprotein synthesis is activated 5 h after a single injection of cortisol and ACTH; a single dose and prolonged administration of cortisol give opposite results. Investigation of the specific radioactivity of apolipoproteins in the blood serum indicates a change in lipoprotein metabolism: disturbance of conversion of very low density into low density lipoproteins. An important role in the pathogenesis of the hyperlipidemia induced by cortisol within the specified period is played not by increased lipoprotein synthesis in the liver, but by a disturbance of their metabolism in the blood.All-Union Cardiological Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Prosented by Academician of the Academy of Medical Sciences of the USSR E. I. Chazov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 538–540, November, 1978.     

Size-dependent in vivo growth potential of adult rat hepatocytes

The present study was performed to determine whether hepatocytes show a size-dependent growth in vivo using as a growth assay system, a retrorsine/partial hepatectomy model of dipeptidyl dipeptidase IV-deficient (DPPIV(-)) mutant Fischer rats. Nearly pure populations of small hepatocytes (SHs) and parenchymal hepatocytes (PHs) were prepared from DPPIV(+) rats. The same number of these SHs and PHs was transplanted into the liver of retrorsine-treated and two-thirds partial hepatectomized DPPIV(-) rats. At 21 days after transplantation, colonies derived from donor hepatocytes were detected as DPPIV(+) cells by enzyme histochemistry. SHs were approximately three times more proliferative than PHs (673 +/- 25 cells/colony versus 226 +/- 10 cells/colony, mean +/- SE). SHs were subfractionated by a fluorescence-activated cell sorter into SH-R2s and SH-R3s. SH-R3s showed a lower extent of granularity and autofluorescence, and a smaller size than SH-R2s that showed characteristics similar to PHs. The growth potential of SH-R3s assayed as above was approximately three times higher than that of SH-R2s (1,101 +/- 46 cells/colony versus 341 +/- 13 cells). These results indicate that the in vivo growth potential of hepatocytes is heterogeneous and is correlated with their size, and the extent of their granularity and autofluorescence.     

星形胶质细胞在凝胶环境下的三维培养

目的:制备不同浓度的三维琼脂糖凝胶以进行星形胶质细胞的体外培养,从而寻找适合细胞生长的理想环境。方法:分别制备1%、2%、3%的琼脂糖凝胶,利用纳米压痕仪测量其弹性模量。星形胶质细胞在凝胶中培养1、3、5、7 d,观察细胞活性以及细胞骨架的变化。结果:随着凝胶浓度的增加,琼脂糖凝胶弹性模量逐渐增加。2%的琼脂糖凝胶的弹性模量最接近筛板组织的弹性模量;在2%、3%琼脂糖凝胶环境下,细胞活性具有较高水平。随着细胞在凝胶中培养的时间增加,星形胶质细胞的突起逐渐伸出,细胞从球形向梭形或星形转变,更接近细胞真实的生长状态。结论:2%琼脂糖凝胶最接近星形胶质细胞在体内的生长环境,细胞成活率较高,是细胞体外三维培养的理想环境。 【关键词】星形胶质细胞;琼脂糖凝胶;三维培养;力学特性     

Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices

To better understand interactions between neurons and extracellular matrix equivalents, embryonic day-18 rat cortical neurons were immobilized and maintained in culture for up to 24 days in agarose and type I collagen gels. Using live/dead staining, neuronal cultures in low density collagen gel lasted at least 3 weeks. At 14 days, over 50% of immobilized cells in collagen gel were found viable while in low density agarose gel no cells survived. In situ cell death detection showed that most, if not all, dead cells in either of the gels underwent apoptosis. The collagen-trapped neurons exhibited normal neuronal polarity and developed long neurites, estimated at over 500 microm. The results suggest that collagen, because it is a major extracellular matrix constituent, suppresses apoptosis and provides a suitable substrate for neuronal survival and differentiation.     

Analysis of defective SV40 DNA by agarose gel electrophoresis.

The SV40 DNA that was generated by undiluted passaging of the virus was analysed by agarose gel electrophoresis. Nine bands of virus DNA were distinguished and each band contained a specific size class of DNA, all shorter than the complete SV40 genome as was determined by electron microscopy measurements. A difference of 2% in length, about 100 base pairs, resulted in a clear band splitting. Two sets of undiluted passaging were established and the defective DNA in the two sets had both different and similar size classes varying in length from 96% to 73% of the unit length SV40 DNA.     

Use of the agarose gel method to identify and quantitate factor VIII:C inhibitors

The agarose gel method for the detection and quantitation of Factor VIII inhibitors was investigated and compared with the Bethesda method. The agarose gel method was standardized for this study by modifications of the original method of Bird. (Bird P: Coagulation in an agarose gel and its application to the detection and measurement of factor VIII antibodies. Br J Haematol 1975; 29:329-340) Precision studies on values obtained by the agarose gel method indicated it is a reproducible assay. Random error was evident in both methods, but variance appeared to be greater in the Bethesda method for most of the samples evaluated. Comparison of the two methods with split patient samples indicated that proportional error is present in the agarose gel method, and that standardization of the method requires additional study. The agarose gel assay detected low levels of inhibitor (down to 3.4 Bethesda units) and all positive samples were identified. A screening procedure modification of the agarose gel method provided the sensitivity to detect Factor VIII inhibitor levels of less than 1.0 Bethesda units. The results indicated that the agarose gel method is a feasible alternative to the Bethesda method for both quantitative and qualitative determination of Factor VIII inhibitors. Furthermore, since reproducibility is better, it may be useful in detecting the "true" fluctuations of inhibitor titers in a given patient.     

Microscope slide electrophoresis of serum lipoproteins in agarose gel

An ultramicro electrophoretic technique for the separation of serum lipoproteins is described using agarose gel on microscope slides.The interaction between the agarose gel and lipoproteins has been reduced and the separation between beta- and pre-beta-lipoproteins has been improved over existing methods. A semiquantitative assay of the lipoproteins can be done in a densitometer. It is suggested that the interaction between agarose gel and lipoproteins, which, without albumin in the system, results in distortion of the lipoprotein bands, may be mediated by free fatty acids.     

Estimation of blood-group-dependent hyperphosphatasemia in healthy subjects by agarose gel electrophoresis

Intestinal alkaline phosphatase (IAP) isozymes in the healthy human sera appears in two isoforms: normal-molecular-weight IAP (NIAP) and high-molecular-weight IAP (HIAP). We have demonstrated that the reference range for ALP activity is higher in blood group B and O secretors than in the other blood groups, for the appearance of these isoforms is depended on blood group B or O secretors. We currently reported that blood groups dependent hyperphosphatasemia in healthy subjects only appeared in blood group B or O secretors. In the present paper, we examined estimation of blood groups dependent hyperphosphatasemia in healthy subjects by use of agarose gel electrophoresis. We classified 104 healthy subjects with the B or O secretors into two groups of blood groups dependent hyperphosphatasemia (n = 5) and normal ALP activity (n = 99). The mean HIAP percentage in blood groups dependent hyperphosphatasemia group (18.5%) was higher than that in normal ALP activity group (9.9%). These results suggested that blood groups dependent hyperphosphatasemia in healthy subjects can be estimated from the HIAP levels by agarose gel electrophoresis.     

Use of immunoglobulin coupled to agarose beads for examining the specificity of conjugates.

Preparations of purified immunoglobulins, light chains, and unrelated proteins (used as controls) were covalently linked to agarose beads and used to study the specificity of fluorescein isothiocyanate conjugates. The data demonstrate how these beads can be used to detect immunological and non-immunological reactivity in conjugates. Commercial conjugates, conjugates prepared in this laboratory, and fluorescein-labeled normal immunoglobulins demonstrated high reactivity with control beads unless chromatographed on diethylaminoethyl-cellulose to select for the proper fluorescein-protein ratio. Undesirable immunological reactivity could be demonstrated in commercial conjugates and was shown to be due to anti-light-chain antibody.     

Effect of tetrahydrocortisol on protein biosynthesis in hepatocytes

Cooperative effect of high-density lipoproteins and glucocorticoids on protein biosynthesis in hepatocytes was associated with the formation of biologically active hormone in Kupffer cells. Apolipoprotein A-I, a constituent of high-density lipoproteins, transported the active hormone from liver macrophages to hepatocytes. Apolipoprotein A-I-induced stimulation of protein biosynthesis was observed only in Kupffer cells. Apolipoprotein A-I alone or in combination with hormones did not change the rate of protein biosynthesis in liver endotheliocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 2, pp. 171–173, February, 2000     

Effect of intravenously administered lipoxygenase metabolites on rat tracheal mucous gel layer thickness

The effect of intravenous injections of 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETE), leukotrienes D4 and E4 (LTD4, LTE4) on tracheal mucous gel layer (TMGL) thickness was assessed in rats. When administered in doses ranging from 0.03 pg to 33 ng per rat, the lipoxygenase metabolites produced significant increases in TMGL thickness. The order of potency of the metabolites was 15-HETE greater than 12-HETE greater than or equal to 5-HETE greater than LTD4 greater than or equal to LTE4. Imidazole (31.6 mg/kg), intravenously, significantly decreased this response. These findings suggest that the mono-HETEs, especially 15-HETE, may be important modulators of airway mucus in the rat.     

A simple immunoblotting method after separation of proteins in agarose gel

A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence of multiple molecular forms of the complement factors C3 and factor B in serum from 2 species, man and chicken, after electrophoretic separation in agarose. We have also demonstrated the usefulness of the method for determining the isoelectric point of proteins after isoelectric focusing in agarose.     

Characterization of adenovirus antibodies by single radial diffusion in agarose gels containing immobilized intact virus particles.

The interaction between antibodies and surface antigens of intact immobilized adenovirus particles was studied by single radial immunodiffusion tests (SRDT) in agarose gels. Purified virus particles and antisera against virus particles of selected serotypes representing different subgroups and against structural components of certain serotypes were employed. Antibodies against hexons, pentons and fibres all gave visible zones. Antisera against the latter components were more efficient in forming zones and this was though to be due to the occurrence of relatively smaller amounts of vertex capsomers and fibre antigen at the virus particle surface. The capacity of antibodies against vertex capsomers to form zones was demonstrated in tests with virus particles of intermediate types. The SRDT with immobilized virus particles was found to be highly sensitive for the demonstration of virus particle surface antigens shared between serotypes. Results obtained in previous studies using virus particles haemagglutination-inhibition (HI) tests including anti-antiserum were confirmed. Cross-reactions mainly occurred within subgroups and were ascribed to a sharing of vertex capsomere antigens. In preliminary studies a rapid typing was achieved by examination of paired human sera from five cases of adenovirus infections.     

Derivation of phenobarbital-responsive immortal rat hepatocytes.

Two lines of rat hepatocytes, designated 6/15 and 6/27, were obtained from carcinogen-treated livers by cultivation in medium containing the liver tumor promoter, phenobarbital (PB). Both lines appeared to be PB-responsive and to have an unlimited in vitro proliferative lifespan, i.e., immortality. The ability of pure 6/27 hepatocytes to form colonies from single cells was strictly dependent upon PB; it was reduced by 97 to 99% in the absence of PB. These hepatocytes were not tumorigenic. For 6/27 hepatocytes in early passages where cultures contained fibroblast contaminants and later when they were a pure culture, PB was able to enhance colony growth from single cells and facilitated population expansion by sustaining DNA synthesis and by inhibiting cell lysis. The 6/15 line displayed PB-dependent colony formation and was not tumorigenic at early passages. At later passages 6/15 hepatocytes were less dependent on PB for colony formation, and they formed hepatocellular carcinoma when transplanted into livers of syngeneic rats. The demonstration that PB sustained the proliferation and viability of hepatocytes with enhanced growth capacity and indefinite proliferative lifespan suggests that PB may be necessary for progression of these chemically initiated hepatocytes to immortal and tumorigenic lines in vitro.     

Effect of complexes of apolipoprotein A-I with tetrahydrocortisol and pregnenolone on protein biosynthesis in rat hepatocytes culture

Complexes of apolipoprotein A-I with tetrahydrocortisol and pregnenolone exhibit high biological activity and increase the rate of protein biosynthesis in the culture of rat hepatocytes. An important role in this process is played by reduced Δ⁴-3-keto group in the A-ring of steroid hormones. A complex of apolipoprotein A-I and pregnenolone modulated the rate of protein biosynthesis in liver cells. Hence, the observed changes are not organ-specific for this steroid. Our results suggest that this mechanism of regulation play an important role in intracellular regeneration and proliferation. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 264–266, September, 2007     

A case of hyper-HDL-cholesterolemia presenting peculiar lipoprotein patterns in agarose gel electrophoresis

Lipoprotein metabolism was analyzed in a patient with marked hyper-HDL-cholesterolemia. A 50 year old male with no symptom of ischemic heart disease or xanthoma had a serum cholesterol level between 293 and 410 mg/dl, and a markedly elevated, HDL-cholesterol level (160-190 mg/dl). The cholesterol content of ultracentrifugally separated HDL2 was exclusively increased, while it was normal in the HDL3 fraction. Analytical ultracentrifugation and HPLC revealed that HDL particles became remarkably larger than the control and, on the contrary, LDL particles became smaller. LPL and LCAT activities were higher in this case, but H-TGL activity was normal. Agarose gel electrophoresis of lipoproteins showed an abnormal broad band which was located between alpha and pre beta band. Serum levels of apolipoprotein A-I, A-II, C-II, C-III and E were higher, while apolipoprotein B level was slightly lower than the control. Cholesteryl ester transfer protein (CETP) activity was demonstrated to be completely deficient in this case, as determined in 10 microliters serum using [3H] CE-labeled HDL3 as donor and VLDL + LDL fraction as acceptor. Since CETP was considered to catalyze the cholesteryl ester transport from HDL to VLDL and LDL, the deficiency of this activity might be the cause of the marked hyper-HDL-cholesterolemia in this patient.     

Deacylation of bacterial lipopolysaccharide in rat hepatocytes in vitro.

The possible role of liver parenchymal cells in the uptake and degradation of bacterial lipopolysaccharide (LPS) was investigated in vitro by employing radiolabelled LPS as substrate. Hepatocytes obtained from Wistar rats by collagenase treatment were found to take up LPS only when it was not linked to the polysaccharide of O-antigen. The amount of LPS taken up increased with time and after 48 h incubation it increased in a dose-dependent manner up to at least 30 micrograms. When incubated with LPS radiolabelled exclusively in the fatty-acid moiety, cultured hepatocytes released lipophilic materials into the culture medium. These were identified as beta-hydroxytetradecanoic acid and triglyceride, in the ratio of 7:I. These results indicate that the R-form of LPS which lacks the O-antigen polysaccharide is taken up and deacylated in hepatocytes, and the derived fatty acids are released into the culture medium either in the free form or after conversion to triglyceride.