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Results for “Direct detection”

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At least 19 recordsLinked to original sources
PubMed1979

Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.

Animals↗
PubMed2025

Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells.

Base editors create precise nucleotide changes in DNA, but their off-target activity remains challenging to quantify. Here, we develop and deploy a direct, in cellulo sequencing assay that simultaneously measures both Cas9-mediated unwinding and deaminase editing of genomic DNA (beCasKAS). Our strategy nominates >460-fold more potential off-target sites than other methods by enriching for Cas9-dependent R-loops immediately preceding editing. Using beCasKAS in primary human T-cells, we observe that mRNA-encoded ABE8e and PAMless ABE8e-SpRY base editors have distinct off-target profiles that can be mitigated by optimizing mRNA dose. Finally, we combine beCasKAS with base-resolution deep learning models to risk-stratify off-target edits by their likelihood of epigenetic dysregulation. Collectively, beCasKAS offers a sensitive and facile tool to optimize the balance between base editor on- and off-target activity.

Journal Article↗
PubMed1976

Beta-adrenergic receptors in rat myocardium: direct detection by a new fluorescent beta-blocker.

A new fluorescent beta-blocker, 9-amino-acridin propranolol (9-AAP), was administered i.v. to rats. Multiple fluorescent 9-AAP binding sites were observed on cardiac muscle cells in frozen sections. Intensity and density of cardiac 9-AAP fluorescence were markedly reduced following pretreatment with (+/-)- and (-)-propranolol but not with (+)-propranolol. Our findings suggest that 9-AAP may label beta-adrenergic receptor sites in rat myocardium.

Acridines↗
PubMed1979

An approach to direct detection of sinus nodal activity in man.

We are reporting a series of 13 out of a total of 16 patients in whom sinus nodal activity was recorded with the use of intra-atrial electrodes, during diagnostic His-bundle electrocardiography investigations. The protocol consisted of the simultaneous monitoring of the following intra-atrial points: high right atrium, low lateral right atrium, left atrium and the His-bundle electrogram (HBE), which accompanied the detecting sonde placed as a loop in the superior vena cava, with the electrodes facing the sinus nodal area. This permitted an adequate assessment of the sequence of the depolarizing activity. All patients showed sinus nodal potentials preceding the initiation of the P-wave in the surface-EKG or the atrial potential in the high right atrial lead, respectively, in the range of 60 to 270 msec depending upon the functional status of the sinus node in each patient. Such waves were clearly separated from the initiation of the atrial potential by an isoelectric line and showed functional traits compatible with the S-A nodal functions, as many authors have previously reported in experimental animal models. The text contains a review of the relevant literature and discussion upon the diagnostic significance of this method in the assessment of sinus nodal disease.

Adult↗
PubMed1979

[The direct detection of sinus nodal potentials in man by means of the signal-averaging technique (author's transl)].

In 13 patients in the age range of 34 to 70 years (mean: 53 +/- 10 years), during diagnostic His-bundle investigations, registrations from the sinus nodal area were analyzed with a signal-averaging computer. In 12 patients we were able to get reproducible per-P-potentials with a sino-atrial conduction interval between 56 and 227 ms. The normal sequence of the excitation process from the atrium to the AV node together with the bundle of His and to the ventricles in all patients, and the reproducibility of the signal-averaged pre-P-potentials, which were well outlined from the atrial potential with a reasonable conduction interval, suggest that these signal-averaged pre-P-potentials reflect the sinus nodal activity rather than an ectopic focus. In 5 of 11 comparable cases there was a good correlation between the signal-averaged sino-atrial conduction intervals and those calculated with the extrastimulus technique. In 2 patients this correlation was moderate. The signal-averaging process may be impaired by coarse random noise, frequent atrial or ventricular extrasystoles, and by fluctuations of the baseline of the sinus nodal lead. The fact that the registrations are performed during normal undisturbed sinus rhythm offers a considerable advantage over the extrastimulus technique.

Action Potentials↗
PubMed1976

Rheumatoid factor-producing cells detected by direct hemolytic plaque assay.

Lymphocytes secreting anti-IgC antibodies, rheumatoid factors (RF), can be detected in the peripheral bloods, synovial fluids, and bone marrows of patients with seropositive rheumatoid arthritis by using a direct plaque-forming cell (PFC) assay with sheep erythrocytes sensitized with reduced and alkylated rabbit IgG hemolysin. The autospecific nature of the RF produced by RF-PFC was indicated by inhibition studies in which the order of patency was human IgG greater than rabbit IgG greater than bovine IgG. In metabolic studies puromycin, cycloheximide, and venblastine suppressed RF-PFC. Cyclic AMP and cyclic GMP were without effect. A need was recognized for using full tissue culture media during the cell separation and plaquing procedures to optimize detection of the RF-PFC. RF-PFC may appear in the blood of patients intermittently despite their continuing presence in the bone marrow. They have been found in the peripheral blood, especially during acutely exacerbating polyarticular synovitis, generalized vasculities, or generally active, aggressive disease. RF-PFC were found in synovial effusions of new or recrduescent acute synovitis. RF-PFC were observed to disappear from the peripheral circulation and the bone marrow during therapy with cytotoxic drugs. The data are consistent with the hypothesis that the appearance of RF-PFC in the peripheral blood represents an anamnestic response to transiently appearing antigen. The nature of the antigen is not specified. The bone marrow may be a site of origin of RF-PFC.

Antibody Specificity↗
PubMed1978

Detection by direct immunofluorescence of antibodies to Treponema pallidum in the cutaneous infiltrates of rabbit syphilomas.

By direct immunofluorescence, with use of fluorescein-labeled sonified Treponema pallidum, specific antibodies were detected in the tissue infiltrates of cutaneous syphilomas in rabbits. Specimens from cutaneous and mucosal inflammatory lesions induced by intradermal injection of a keratinous substance from an epidermal cyst served as controls. Granular fluorescence was detected in the dermis of 11 of the 12 syphiloma specimens and corresponded to areas of heavy plasma cell infiltrates, and some fluorescence was found directly on plasma cells identified by subsequent staining with hematoxylin and eosin. This fluorescence could be blocked by preabsorption. Control slides did not show any fluorescence. Immunopathologic techniques using labeled antigens may be of diagnostic value in syphilis and other infectious disorders which feature specific infiltrates.

Animals↗
PubMed1975

A hemolytic plaque assay for the detection of direct and indirect antibody-forming cells to keyhole limpet hemocyanin.

A procedure is described for the in vitro enumeration of individual lymphoid cells producing antibody against Keyhole Limpet Hemocyanin (KLH) by a modification of the hemolytic plaque technique. Optimal conditions for the coupling of KLH to sheep erythrocytes by chromic chloride are described. Plaque-forming cells are detected in a liquid monolayer slide chamber assay system. The kinetics of the in vivo primary and secondary PFC responses of both rabbit popliteal lymph node cells and mouse spleen cells are described. Both direct (IgM) and indirect (IgG) plaque-forming cells can be enumerated. The method can also be used to detect an in vitro anamnestic response in dispersed rabbit lymph node cell suspensions. The method is simple, extremely sensitive and the results correlate with those previously obtained in which newly synthesized antibody was detected in similar systems using the coprecipitation assay.

Animals↗
PubMed1977

High performance liquid chromatographic separation and direct ultraviolet detection of phospholipids.

A fast and efficient method for the separation of (phospho)lipids by high performance liquid chromatography using n-hexane, 2-propanol, water mixtures as the solvent system is described. The lipid separation occurs on a LiChrosorb Si-60 (10 micron) column and the individual components are monitored directly by ultraviolet absorption at 206 nm. Of a total lipid extract from erythrocytes a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are only partly separated under these circumstances. Furthermore, a mixture of synthetic phospholipids, i.e. 1,2-dilauroyl-sn-glycero-3-phosphatidic acid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-1'-sn-glycerol and 1,2-dioleoyl-sn-glycero-3-phosphocholine has been completely resolved. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some cases, as is shown for 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine and 1,2-didocos-13'-cis-enoyl-sn-glycero-3-phosphocholine.

Animals↗
PubMed1979

Demineralization in cystic fibrosis detected by direct photon absorptiometry.

Bone mineral content, bone width, and their ratio were measured in patients with cystic fibrosis (CF) using monoenergetic photon absorptiometry. Serial measurements of the radius and ulna were made in 27 patients with CF and were compared with 968 age-matched controls. Demineralization was found in 37% of the boys and 63% of the girls. Patients under age 10 years had normal bone mineral content and nine of 15 patients aged 13 or older were demineralized (P less than .01). Demineralization correlated with the extent of weight reduction in patients (P less than .001). Patients most likely to be demineralized were adolescent girls. To our knowledge, this is the first report of bone mineral status of children with CF, and the results indicate that a sizable proportion of these patients may be demineralized without overt rickets.

Adolescent↗
PubMed1977

Direct immunochemical detection of prostaglandin-E and cyclic nucleotides in human malignant tumors.

Immunofluorescent localization of prostaglandin-E (PGE), cyclic AMP (CAMP), and cyclic GMP (cGMP) was studied in tumor tissues from 40 patients with a variety of solid tumors. Representative normal tissues served as controls. Rabbit antisera specific for PGE or the cyclic nucleotides were used, and the reactions observed were correlated with the degree and type of lymphocytic reaction at the tumor margins. Strong PGE immunofluorescence was detected in tumor cells in 27 of 42 malignancies; by contrast nine of 13 normal tissues showed weak PGE reactions, cAMP was detected in 30 of the 42 malignancies; cGMP was noted in only seven of the 42 malignant tissues and in none of the normal tissues studied. The most common malignant tumor profile (17/42) was that of positive PGE and cAMP and negative cGMP staining. Tumors showing strong staining with anti-PGE or cAMP demonstrated a distinct trend towards heavier lymphocytic infiltration with a predominance of T cells at their margins, although this association did not reach statistical significance in the present material.

B-Lymphocytes↗