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At least 289 records · Page 16Linked to original sources
PubMed1988

Lymphocyte-virus interactions. Identification of a restriction fragment permitting virus replication in lymphocytes.

We are interested in understanding the effects of virus infection on lymphocyte function. To approach this question, we used a unique system of two genetically related leporipoxviruses to generate recombinants. One of these viruses, malignant fibroma virus (MV), replicates in many different cell types, including lymphocytes. The other, Shope fibroma virus (SFV), replicates principally in fibroblasts, but cannot replicate in lymphocytes. Fibroblasts infected with SFV received restriction fragments from MV by transfection. Recombinant viruses were selected in vitro for their ability to replicate in lymphocytes. By these means we have identified one restriction fragment, the 10.8-kb BamHI "C" fragment, capable of transferring from MV to SFV the ability to replicate in lymphocytes. A family of recombinants bearing different sized inserts of this restriction fragment has been isolated and is being characterized. Lymphocytotropic recombinants bearing portions of this restriction fragment produce colony morphology in vitro intermediate between MV's plaques and SFV's foci. On the basis of their ability to grow in and suppress mitogen responsiveness of lymphocytes, these recombinants may be classified into four different groups. Group 1 viruses are the most immunosuppressive, whereas those of group 4 are least. These traits correlate with ability to replicate in lymphocytes. Genetic analysis of recombinants indicates that the most immunosuppressive recombinants do not necessarily contain the most fragment C DNA. Therefore, we have identified a restriction fragment, one or more of the genes of which are sufficient to allow an otherwise nonlymphocytotropic virus to replicate in lymphocytes. Additional genetic and immunologic analysis should permit us to determine the structure and function of the protein responsible for this effect.

Animals↗
PubMed1987

Leucyl-leucine methyl ester treatment of donor cells permits establishment of immunocompetent parent----F1 chimeras that are selectively tolerant of host alloantigens.

Treatment of murine lymphocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte precursors, and the capacity to cause lethal graft-vs-host disease, whereas bone marrow stem cell function and alloantigen-induced L3T4+ T helper function remains intact. The present studies assess the immunocompetence of allogeneic bone marrow chimeras established by reconstituting irradiated (C57BL/6 X DBA/2)F1 (B6D2F1) mice with Leu-Leu-OMe-treated C57BL/6 (B6) bone marrow and spleen cells. Spleen cells from such chimeras were found to have normal B and T cell mitogenic responses. Furthermore, levels of natural-killer cell function were comparable to those observed in B6----B6 syngeneic radiation chimeras established without Leu-Leu-OMe treatment of donor cells. Spleen cells from B6----B6D2F1 mice were identical with B6----B6 or B6 mice in allostimulatory capacity and thus contained no discernible cells of non-H-2b phenotype. Whereas B6----B6D2F1 spleen cells demonstrated alloproliferative and allocytotoxic responses toward H-2k bearing spleen cells, no H-2d specific proliferative or cytotoxic responses could be elicited. B6----B6D2F1 spleen cells did not suppress the generation of anti-H-2d or anti-H-2k proliferative or cytotoxic responses from control B6 spleen cells. Furthermore, addition of rat concanavalin A supernatants did not reconstitute anti-H-2d responses of B6----B6D2F1 chimeric spleen cells. Thus, Leu-Leu-OMe treatment of B6 donor cells not only prevents lethal graft-vs-host disease, but also permits establishment of long-lived parent----F1 chimeras that are selectively tolerant of host H-2 disparate alloantigens, but fully immunocompetent with respect to natural killer cell function, B and T cell mitogenesis, and anti-third party alloresponsiveness.

Animals↗
PubMed1985

Quantification of pulsatile flow during cardiopulmonary bypass to permit direct comparison of the effectiveness of various types of "pulsatile" and "nonpulsatile" flow.

The relative merits of adding a "pulsatile" component to flow during cardiopulmonary bypass (CPB) has long generated controversy, the resolution of which has been hampered by lack of quantification of the "pulsatility" delivered by different devices. The present experimental series had two goals: to quantify the "pulsatility" of blood flow during CPB in terms of pulse rate and pulsatility index (PI) and to examine which aspects of a "pulsed flow" provide clinical benefits. A flow waveform can be expressed in terms of its baseline rate and its PI, the sum of the square of its harmonics components divided by the square of the mean flow. We used PI to quantify the pulsatility of blood flow in the descending thoracic aorta and used changes in the serum lactate level as an indication of end organ flow. In one experimental series seven adult mongrel dogs were placed on roller pump CPB at a constant flow of 100 ml/kg/min. After a 20-minute stabilization period a roller pump wave and three different pulse shapes (generated by a computer-controlled hydraulic pump) were evaluated for 15 minutes each. The pulse wave shapes were graded, with C being the sharpest and A the least sharp. In a second series six other dogs were placed on CPB and were subjected to roller pump perfusion and three pulse waves of identical shape but at different rates. The results indicated that a combination of a minimum PI of 1.88 and a minimum rate of 80 bpm were necessary to significantly reduce lactate production as compared with roller pump perfusion. Thus the same mean flow can have very different physiologic effects depending on how it is delivered. This quantification method permits direct comparison of different "pulsatile waveforms" and provides a means for identification of optimal pulsatile flow.

Animals↗
PubMed1983

Peptide intermediates in the degradation of cellular proteins. Bestatin permits their accumulation in mouse liver in vivo.

We have previously shown that bestatin (an aminopeptidase inhibitor) permits the accumulation of di- and tripeptide intermediates in the degradation of abnormal globin (Botbol, V., and Scornik, O. A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 710-713; Botbol, V., and Scornik, O. A. (1979) J. Biol. Chem. 254, 11254-11257). We now report that this drug (1 mg, intravenously, 5-30 min) causes similar intermediates to appear during breakdown of normal cellular proteins in the livers of live mice labeled 1 or 20 h before with L-[1-14C]Leu or L-[1-14C]Arg. These intermediates represent an estimated 20% of all degraded cellular protein. Lysosomal degradation of labeled asialoglycoproteins taken up by endocytosis is less affected by bestatin. After homogenization and centrifugation of the livers, we find a major fraction of the intermediates in the cytosol. Another fraction sediments at 27,000 X g in 15 min. The fraction that sediments is larger for Arg-labeled (30%) than for Leu-labeled (10%) peptides. The particulate fraction does not represent soluble peptides trapped in the pellet during fractionation because it does not appear when soluble intermediates are added to nonradioactive homogenates. The presence of intermediates in particulate and soluble fractions could result from protein breakdown either in both compartments or in organelles from which the peptides escape. We favor the latter possibility because (a) the particulate fraction does not increase after stimulation of autophagy by injection of glucagon (40 micrograms, 75 min before killing), (b) the subcellular distribution is the same whether intermediates are produced during the degradation of short or long lived proteins, and (c) chromatographic fingerprints of the particulate and soluble components reveal the same seven bands. The presence of well defined intermediates of cellular protein degradation in the particulate fraction of liver homogenates provides a valuable marker in the isolation and characterization of autophagic organelles.

Aminopeptidases↗
PubMed1982

A simple modification of Greiner electronics selective analyser G300 permitting use of capillary blood samples.

A simple modification of Greiner electronics selective analyser G300 allowing direct transference of serum samples to the process tubes is described. The modified instrument permitted use of pediatric and capillary blood samples. Eight different capillary analyses were tested on the modified instrument and the results agreed with those obtained by the ordinary analytical macroprocedures of the instrument.

Automation↗
PubMed1981

[Does echocardiography Tm permit to determine the true contractile state of the left ventricle of hemodialysis patients? (author's transl)].

M. mode echocardiography was performed on 16 chronic hemodialysis patients (12 men, 4 women, average use of 21, hematocrit around 24 +/- 5%) with a normal blood pressure and no clinical or roentgenographic signs of heart failure, 18 to 22 hours after the end of a dialysis. Renal diseases due to hypertension, diabetes or amyloidosis were excluded from the study. 8 normal subjects of similar age, heart rate and blood pressure were used as a test group. On these 24 persons, and diastolic time diameter index (DTDI) and end systolic time diameter index (STDI), ejection time (ET), mean velocity of circumferential fiber shortening (VCF) and ejection fraction were calculated. DTDI of hemodialysis patients (31 mm/m(2) +/- 2) is greater than DTDI of the control subjects, and STDI and ET are the same. This explains the increase of VCF (1,69 +/- 0,10 c/s) and EF (0,78 +/- 0,05). After a three minute compression of the fistulas the differences disappear. These results suggest that the previous exam conditions permit a better determination of the true contractile state of the left ventricule of hemodialyzed patients if one disregards the load changes due to the fistula, anemia and the intermittent volume expansion.

Adolescent↗
PubMed1993

A paraffin wax technique of Golgi-Cox impregnated CNS that permits the joint application of other histological and immunocytochemical techniques.

Our technique permits the application of other histological and immunohistological methods to sections of CNS already impregnated in a modified Golgi-Cox solution. This combined approach gives a more comprehensive picture of anatomical, cytological and antigenic relationships. Furthermore the technique is reproducible, sections are of known thickness and there is no problem with long term storage. Details of the technique are given and its application in neuroanatomy and neuropathology is discussed.

Brain↗
PubMed1994

Hereditary tyrosinemia type I: strong association with haplotype 6 in French Canadians permits simple carrier detection and prenatal diagnosis.

Hereditary tyrosinemia type 1 (HT1), a severe inborn error of tyrosine catabolism, is caused by deficiency of the terminal enzyme, fumarylacetoacetate hydrolase (FAH). The highest reported frequency of HT1 is in the French Canadian population, especially in the Saguenay-Lac-St-Jean region. Using human FAH cDNA probes, we have identified 10 haplotypes with TaqI, KpnI, RsaI, BglII, and MspI RFLPs in 118 normal chromosomes from the French Canadian population. Interestingly, in 29 HT1 children, a prevalent haplotype, haplotype 6, was found to be strongly associated with the disease, at a frequency of 90% of alleles, as compared with approximately 18% in 35 control individuals. This increased to 96% in the 24 patients originating from Saguenay-Lac-St-Jean. These results suggest that one or only a few prevailing mutations are responsible for most of the HT1 cases in Saguenay-Lac-St-Jean. Since most patients were found to be homozygous for a specific haplotype in this population, FAH RFLPs have permitted simple carrier detection in nine different informative HT1 families, with a confidence level of 99.9%. Heterozygosity rate values obtained from 52 carriers indicated that approximately 88% of families at risk from Saguenay-Lac-St-Jean are fully or partially informative. Prenatal diagnosis was also achieved in an American family. Analysis of 24 HT1 patients from nine countries gave a frequency of approximately 52% for haplotype 6, suggesting a relatively high association, worldwide, of HT1 with this haplotype.

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli.

A new phagemid vector, pSIT, was constructed that allows both oligonucleotide-directed mutagenesis and tightly controlled, high-level expression of proteins in Escherichia coli. An efficient rate of mutagenesis is achieved by taking advantage of the double oligonucleotide primer technique. In addition to the mutagenic primer, a second oligonucleotide primer conferring antibiotic resistance to the mutant DNA strand is annealed to single-strand DNA. Selection for the antibiotic thus increases the frequency of mutants. High-level and tightly controlled expression of heterologous proteins is enabled by utilizing a very strong hybrid T7lac promoter and lac repressor in conjunction with T7 RNA polymerase, as well as a high copy number of the vector. The pSIT phagemid permits overexpression of proteins and their mutants without subclonings from mutagenic to expression constructs, which saves time, especially when multiple mutations of the same protein are proposed. A retroviral proteinase precursor, toxic for E. coli, was successfully expressed to a high level, and a series of mutants of this protein was readily obtained.

Amino Acid Sequence↗
PubMed1994

Posttranscriptional regulation of Id1 activity in cardiac muscle. Alternative splicing of novel Id1 transcript permits homodimerization.

The transcriptional regulatory protein Id, a negatively trans-acting protein with a helix-loop-helix motif that is expressed in many proliferating tissues early in development, continues to be expressed in postmitotic adult cardiac myocytes and vascular smooth muscle. Following the observation of a "doublet" band of 1.1 and 1.25 kilobases on Northern hybridizations of Id1 cDNA with mRNA isolated from both cardiac muscle and vascular smooth muscle cells, we identified and sequenced an alternatively spliced Id1 gene product containing an insert of 214 base pairs within the coding domain of the original Id1 cDNA. A protein with a molecular mass corresponding to that predicted by the Id1.25-kilobase mRNA sequence could be identified on immunoblots of cell lysates from neonatal and adult rat ventricular myocytes. The insert appears to be a "coding intron," based on the presence of intron-exon consensus sequences at the insert boundaries and the presence of the originally described Id1 carboxyl-terminal coding sequence immediately downstream from, and out of frame with, this insert. In contrast to Id1 and Id2, which do not form homodimers, the carboxyl-terminal sequence of this alternatively spliced Id1 transcript, termed Id1.25, permits homodimerization. Thus, alternative splicing of Id1 may allow for tissue-specific expression of Id1, while formation of homodimers could provide a post-translational mechanism to regulate the ability of Id1.25 to bind and inactivate E2A gene products.

Alternative Splicing↗
PubMed1993

The adenovirus-mediated delivery of a reporter gene permits the assessment of androgen receptor function in genital skin fibroblast cultures. Stimulation of Gs and inhibition of G(o).

Defects in the androgen receptor cause a spectrum of abnormalities in genetic males ranging from phenotypic women with testicular feminization to men with minor defects in fertility and/or virilization. The diagnosis of androgen resistance can be quite cumbersome, including analysis of the family history, karyotyping, endocrine studies, measurement of androgen binding in genital skin fibroblasts, and, in some instances, sequencing of mutant cDNAs. Furthermore, androgen-binding studies may be normal in patients with qualitative receptor abnormalities or mutations in the DNA-binding domain of the receptor. To circumvent these difficulties, we have used a recombinant adenovirus to deliver an androgen-responsive reporter gene (mouse mammary tumor virus-luciferase) to fibroblasts cultured from genital skin from 12 normal controls and from eight individuals with complete testicular feminization. Following incubation with androgen (2 nM mibolerone) for 72 h, luciferase activity in normal fibroblasts increased > 10-fold (range 11-200-fold) in a manner that corresponded with the level of androgen receptor detected in ligand-binding assays. By contrast, luciferase activity increased negligibly in fibroblasts from individuals with testicular feminization (average = 1.2-fold increase). This assay permits a direct assessment of endogenous androgen receptor function in cells and should be a powerful aid in the diagnosis of androgen resistance.

Adenoviridae↗
PubMed1996

A1, a Bcl-2 family member, prolongs cell survival and permits myeloid differentiation.

A1, a bcl-2 family member, has been identified as a hematopoietic-specific, early inducible gene. In this study it is shown that stable transfection of A1 into an interleukin-3 (IL-3)-dependent myeloid precursor cell line, 32D c13, leads to a retardation of IL-3 withdrawal-induced cell death similar to that observed with transfection of bcl-2. However, unlike bcl-2. A1 expression permits the accumulation of differentiated myeloid cells both before and after IL-3 withdrawal. Total cell accumulation, on the other hand, is considerably greater after IL-3 deprivation in the bcl-2 transfectant than in A1-expressing cells. Cells cotransfected with the two genes behave similarly to cells singly transfected with bcl-2, except that viability following IL-3 withdrawal is somewhat further enhanced. These results suggest that these two proteins have distinct roles that may be related to the divergent regulation of their expression during myeloid differentiation.

Animals↗
PubMed1996

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.

Adenosine Triphosphate↗