Search PubMed⌕ Search

Biomedical subjects

D A Jackson

Publications and source records attributed to D A Jackson.

At least 109 records · Page 6Linked to original sources
PubMed1988

Conditional enhancement of liver-specific gene transcription.

We sought to develop a cell line in which liver-specific transcription could be induced at will, to facilitate the study of factors that cause hepatocyte-specific transcription of the serum albumin gene in mice. We therefore created the H2.35 cell line from mouse hepatocytes infected with a temperature-sensitive strain of simian virus 40. During routine propagation at the permissive temperature, H2.35 cells exhibit extremely low levels of albumin transcription and mRNA. Albumin mRNA increases at least 100-fold when H2.35 cells are cultured at the restrictive temperature and in serum-free medium on a collagen substratum; the two latter conditions maintain the differentiated state of primary hepatocyte cultures. Although a major cause of the mRNA increase is posttranscriptional, the transcription rates of albumin and other liver-specific genes increase significantly. Transient-transfection experiments demonstrated that an induction of transcription is caused by activation of an albumin upstream sequence that was previously shown to enhance liver-specific transcription in transgenic mice. Thus, hepatocyte differentiation appears to be maintained in part by extracellular signals that stimulate the activity of a tissue-specific enhancer element.

Animals↗
PubMed1988

Circadian variation in fat concentration of breast-milk in a rural northern Thai population.

1. Twenty-five northern Thai mothers, breast-feeding their infants on demand, were studied in their homes for 24 h. All breast-feeds were test-weighed and pre- and post-feed expressed breast-milk samples (0.5 ml) taken at each feed. 2. The fat concentration of milk taken during a feed showed significant circadian variation, with maximum values between 16.00 and 20.00 hours and minimum values between 04.00 and 08.00 hours. Fat concentration at the start and at the end of a feed also varied significantly over 24 h. 3. Multiple regression analysis showed that the most important predictor of fat concentration at a feed was the length of time elapsed since the previous feed--the longer this interval, the lower the subsequent fat concentration. Other significant predictors were the fat concentration at the end of the previous feed, and the milk intake at the previous and at the current feed. 4. Fat concentration declined between feeds in proportion to the length of time between feeds, but the decline was less between sleep feeds than between waking feeds. This would appear to be a reflection of the lower post-feed fat concentration and higher pre-feed fat concentration of sleep feeds compared with waking feeds, when other variables relating to feeding pattern are taken into account. 5. The larger the milk intake at a feed, the greater was the increase in fat concentration from the start to the end of the feed. The change in fat concentration was less in feeds taking place during the sleep period than in daytime feeds.

Adult↗
PubMed1988

Estimation of 24 h breast-milk fat concentration and fat intake in rural northern Thailand.

1. The present study assesses the accuracy with which mean 24 h breast-milk composition can be estimated if milk samples can only be collected during the daytime. 2. Twenty-five northern Thai mothers, feeding their infants on demand, were studied in their homes for 24 h. All feeds were test-weighed and 0.5 ml pre- and post-feed expressed milk samples taken at each feed. 3. If daytime sampling was restricted to two breast-feeds, it was found that the best estimate of 24 h fat concentration was given by two randomly chosen daytime feeds, predicting 24 h fat concentration with 95% confidence limits of +/- 7.0 g/l (equivalent to 21% of mean 24 h fat concentration). 4. Alternative sampling methods using the mean fat concentration of the feed after 08.00 hours and after 18.00 hours, or the first two feeds after 12.00 hours, predicted fat concentration with 95% confidence limits of +/- 9.7 g/l and +/- 8.9 g/l (28 and 26% of mean 24 h fat concentration) respectively. 5. If well-tolerated by mothers, it would be preferable to sample all daytime feeds, since this reduces the 95% confidence limits to +/- 3.3 g/l, equivalent to 10% of the mean 24 h fat concentration.

Adult↗
PubMed1988

A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin.

We describe a method for permeabilizing and extracting cells that preserves both structure and function whilst allowing the cell derivatives to be handled freely. Cells are encapsulated in microbeads of agarose; the coat of agarose, which is freely permeable to small molecules, forms a protective layer around fragile cell constituents. Cells are then permeabilized by the non-ionic detergent Triton X-100 or antibody and complement in a buffer whose ionic composition mimics that of the cytoplasm. The resulting structures have been characterized morphologically (by immunofluorescence and electron microscopy) and biochemically. Lysis with Triton removes both cell and nuclear membranes, and extracts most of the cytoplasm to leave chromatin surrounded by cytoskeleton; nucleus and cytoplasm then become accessible to triphosphates, enzymes and antibodies. Lysis with complement permeabilizes the cell membrane but leaves the nuclear membrane intact; triphosphates and restriction enzymes, but not antibodies, can then enter both nucleus and cytoplasm. Both types of lysis yield preparations whose chromatin template remains essentially intact, and which is replicated and transcribed at rates close to, or greater than, those found in vivo. Treatment of complement-lysed cells with Triton reduces the very efficient DNA synthesis, implying that the nuclear membrane is involved, directly or indirectly, in replication.

Cell Nucleus↗
PubMed1988

Infant weight in relation to nutritional intake and morbidity in northern Thailand.

A cross-sectional study of 58 infants (age range 2-53 weeks) was carried out in a rural area of northern Thailand. Total dietary intake (energy and protein from breastmilk and supplementary food) was measured directly during continuous observation over 48 h of randomly selected study subjects in their homes. Associations between infant weight and total energy intake, total protein intake and morbidity were analysed by multiple regression analysis. Up to 6 months of age, heavier infants had larger energy intakes, when age and birthweight were taken into account. In the 7-12 months age group, heavier infants consumed more protein and had fewer respiratory infections. Weaned infants older than 6 months were significantly heavier and consumed more protein than infants of similar age who were still receiving breastmilk. The lower protein intakes of the latter infants were due to the low protein concentration of breastmilk compared with supplementary food.

Body Weight↗
PubMed1987

The automatic sampling shield: a device for sampling suckled breast milk.

The automatic sampling shield uses the negative pressure exerted by the breast-feeding baby during sucking to withdraw a series of small samples from the milk stream. Shield performance was studied at four feeds for each of 13 babies. Acceptance of the shield was good (96% of feeds) and samples were obtained in 88% of feeds. Sample weight was large enough for micro-biochemical analysis of composition (median weight 1.3 g), without depriving the baby of important amounts of milk (median percentage of weight of milk intake was 4.3% and sample weight was less than 5 g in all cases). The shield should sample a constant proportion throughout the feed, in order to obtain a sample that is representative of the infant's nutrient intake. Fractional test-weighing of feeds showed a tendency for a larger proportion of the feed to be collected at slower flow rates. Modelling on the basis of the observed flow profiles in conjunction with a theoretical fat concentration profile showed that the observed 'disproportionality' would have over-estimated fat intake by a mean of 2.6% per feed (S.D.9.1 range -18.9% to + 16.9%). Using the shield in random sequence at two out of the first four feeds after 0800 h, showed that an infant's mean milk intake with the shield was on average 32% less than on control feeds. Allowing for possible compensation by the infant when control feeds followed low shield intakes, mean milk intakes with the shield were 17% smaller than without the shield.

Breast Feeding↗
PubMed1987

Indirect test weighing: a non-intrusive technique for estimating night-time breast milk intake.

Indirect test weighing (ITW), for estimating night-time breast milk intake, is based on separate weighings of mother and infant before and after night-time sleep. Weight changes due to the exchange of milk overnight are reciprocal, so that if all other sources of weight change (eg, urine loss, supplementary fluid intake) are either controlled or measured, the difference between the mother's overnight weight loss and her infant's overnight weight gain represents their combined net evaporative water loss (EWL). The proportion of the combined EWL due to the infant is partitioned out as a function of the relative metabolic body sizes (kWt0.73) of mother and infant, and added to the infant's net overnight weight gain to provide an estimate of night-time breast milk intake. Validation studies, conducted in Northern Thailand, in which ITW was compared with direct test weighing (DTW) at each night feed, are reported for 13 infants over 3 nights at 5 d of age, and for 19 infants over 2 nights at 6 weeks of age or more. The regression equation for estimated milk intake against measured milk intake across all ages was y = 0.988x + 2.75, r = 0.927, n = 69 with 95 per cent prediction intervals of +/- 36 g for a range of intakes of 0-250 g. Rigorous control of data collection and taking account of the infant's age suggest that the prediction intervals for individual estimates can be improved to +/- 18 g at 5 d, and +/- 27 g at 6 weeks and over. Theoretical aspects of the relationship of EWL to body weight are discussed with a view to optimizing the accuracy of the technique.

Body Water↗
PubMed1986

A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin.

An insoluble DNA polymerase activity that replicates the intact chromatin template at 85% of the rate found in vivo has been partially characterized. HeLa cells, encapsulated in agarose microbeads, are lysed using an isotonic salt concentration: the resulting encapsulated nuclei contain polymerase associated with a nucleoskeleton and the unbroken template. This preparation can be manipulated freely without aggregation or breaking the DNA and yet is accessible to enzymes and other probes. The major activity, which is sensitive to aphidicolin, is found only in S-phase nuclei and replicates DNA semi-conservatively, forming intermediates that are ligated efficiently into larger products.

Autoradiography↗
PubMed1986

Different populations of DNA polymerase alpha in HeLa cells.

Three different populations of HeLa DNA polymerase alpha have been distinguished using a novel preparation of chromatin isolated using an isotonic salt concentration, which contains intact DNA. One synthesizes DNA in vitro at 85% of the rate in vivo, is found only in S-phase nuclei tightly associated with the nucleoskeleton and requires unbroken DNA in the form of chromatin as a template: we assume this is the authentic S-phase activity. On incubation at 37 degrees C, this activity dissociates from the nucleoskeleton to give a soluble activity that prefers broken templates. This soluble activity is in turn heterogeneous, containing active complexes of about 0 X 75 X 10(6) and 3 X 10(6) Mr. The third activity is also soluble and released by lysing cells at any stage of the cell cycle. It, too, prefers broken templates. The authentic activity is obscured by the soluble ones if broken templates are provided.

Antibodies↗
PubMed1986

Replication occurs at a nucleoskeleton.

The site of S-phase DNA synthesis has been the subject of recurring controversy. All recent evidence supporting a site fixed to some nuclear sub-structure is derived from studies in which cells or nuclei have been extracted in hypertonic salt concentrations. The controversy centres on whether the resulting nuclear matrices or cages have counterparts in vivo or are simply artefacts. Using isotonic conditions throughout the isolation and analytic procedures we have now reinvestigated the site of replication. Cells are encapsulated in agarose microbeads and lysed to leave encapsulated nuclei which are nevertheless completely accessible to enzymes. After incubation with endonucleases, most chromatin can be electroeluted from beads: however, nascent DNA and active DNA polymerase remain entrapped. Since chromatin particles containing DNA the size of 125 kbp can electroelute, we conclude that the polymerizing complex is attached to a nucleoskeleton which is too large to escape. We have also studied various artefacts induced by departure from isotonic conditions. Perhaps surprisingly, the hypotonic conditions used during isolation of nuclei by conventional procedures are a significant source of artefact.

Cell Division↗
PubMed1985

A general method for preparing chromatin containing intact DNA.

A simple and general method is described for preparing chromatin from eukaryotic cells using isotonic conditions. First, cells are encapsulated in agarose microbeads and then lysed using Triton X-100 in the presence of a chelating agent and a physiological concentration of salt. Most cytoplasmic proteins and RNA diffuse rapidly out through pores in the beads to leave encapsulated chromatin which is nevertheless completely accessible to enzymes and other probes. This chromatin can be manipulated freely without aggregation in a variety of different salt and detergent concentrations. It also contains intact DNA since removal of the histones releases superhelical DNA. Conditions are described for incubating this chromatin at 37 degrees C in the presence of Mg2+ ions without any nicking of the DNA. We illustrate the usefulness of this chromatin in investigations on the attachment of nascent RNA to the nucleoskeleton, the accessibility of the ribosomal locus to EcoRI and the properties of the endogenous RNA polymerase II. This type of chromatin preparation should prove useful for both structural and functional studies.

Cell Nucleus↗
PubMed1985

Transcription occurs at a nucleoskeleton.

Native chromatin aggregates under isotonic conditions so it is generally studied using higher or lower salt concentrations. This has led to different interpretations of how transcription might occur. Studies using hypertonically-isolated preparations suggest that DNA functions in close association with a skeletal nuclear substructure, the matrix or cage, but such a structure is not usually seen under hypotonic conditions (e.g., in 'Miller-spreads'). Using a novel method for preparing chromatin under isotonic conditions we have investigated the site of transcription. We find that all three constituents of the transcription complex, nascent transcripts, active RNA polymerase and genes being transcribed are all closely associated with some structure too large to be electroeluted from the nucleus. Hypotonic treatment partly disrupts this association. We suggest a model for transcription that involves the participation of a nucleoskeleton at the active site and reconcile the contradictory results obtained using different salt concentrations.

Cell Nucleus↗
PubMed1985

Nadolol in combination with indapamide and xipamide in resistant hypertensives.

Twenty-four hypertensive patients have been studied. All had blood pressure recordings greater than 160/95 mmHg on 3 occasions whilst taking a beta blocker and two other antihypertensive agents in therapeutic doses. Compliance was checked by intermittent urine analysis for the relevant beta-blocker. These difficult to control hypertensives were treated with nadolol alone, nadolol plus indapamide and nadolol plus xipamide each for 2 months in random order. The aim was to reduce the blood pressure to below 160/95 mmHg. The supine blood pressure on nadolol alone (167/100 mmHg) was comparable to that on the previous three drug regimens (157/100 mmHg), the other two treatments were more effective (145/90 and 148/93 mmHg respectively). Hypokalaemia (serum potassium below 3.5 mmol/l) occurred in six individuals but occurred more frequently on xipamide than on indapamide.

Adult↗
PubMed1984

Attachment of repeated sequences to the nuclear cage.

Nuclear DNA is probably organized into loops by attachment to a sub-structure in vivo. When HeLa cells are lysed in Triton and 2M NaCl the resulting nucleoids contain naked DNA which is supercoiled so the loops must remain intact. We have attempted to identify sequences responsible for attaching these loops to the nuclear sub-structure by progressively detaching DNA with various nucleases. Fragments at the 5' end of the ribosomal RNA locus, and a variety of transcribed and repeated sequences, are shown to lie relatively close to attachment points. This implies that sequences cannot be arranged randomly. However no "attachment sequence" could be identified.

Base Sequence↗
PubMed1984

Replication and transcription depend on attachment of DNA to the nuclear cage.

When living cells are lysed in a non-ionic detergent and 2 M-NaCl, structures are released that resemble nuclei. They contain naked nuclear DNA packaged within a flexible cage of RNA and protein. Since the DNA is supercoiled, it must be intact and looped by attachment to the cage. It is argued that this cage is the active site of the key nuclear functions, transcription and replication: outlying sequences are activated by attachment to polymerases at the cage. This thesis is supported by the close and specific association of nascent RNA with cages, the attachment of active viral sequences (in transformed and productively infected cells) and the attachment of nascent DNA during both normal and repair synthesis.

Cell Nucleus↗
PubMed1983

Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves.

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.

Journal Article↗