Localization of DNA sequences required for human centromere function through an analysis of rearranged Y chromosomes.

We have localized the DNA sequences required for mitotic centromere function on the human Y chromosome. Analysis of 33 rearranged Y chromosomes allowed the centromere to be placed in interval 8 of a 24-interval deletion map. Although this interval is polymorphic in size, it can be as small as approximately 500kb. It contains alphoid satellite DNA and approximately 300kb of adjacent Yp sequences. Chromosomes with rearrangements in this region were analysed in detail. Two translocation chromosomes and one monocentric isochromosome had breakpoints within the alphoid array. Of 12 suppressed Y centromeres on translocation chromosomes and dicentric isochromosomes that were also analysed two showed deletions one of which only removed alphoid DNA. These results indicate that alphoid DNA is a functional part of the Y chromosome centromere.

Structure of the pericentric long arm region of the human Y chromosome.

We have analysed the sequence organization of the DNA in the pericentric region of the long arm of the human Y chromosome. The structures of one cosmid and three yeast artificial chromosome clones were determined. The region consists of a mosaic of the known 5, 48 and 68 base-pair tandemly repeated sequences and at least five novel repeated sequence families. A long range-map of approximately 3.5 x 10(6) base-pairs of genomic DNA was constructed that placed the clones between about 500 x 10(3) and 850 x 10(3) base-pairs from the long arm edge of the centromeric alphoid DNA array.

Structure of the sequences adjacent to the centromeric alphoid satellite DNA array on the human Y chromosome.

Eighteen yeast artificial chromosome (YAC) clones containing alphoid satellite DNA and adjacent sequences from the human Y chromosome have been identified from three different YAC libraries. Restriction site mapping of the genomic alphoid arrays and the YACs has allowed seven of the alphoid clones to be positioned on the arrays. Three clones extend into flanking sequences. At one edge the alphoid DNA is highly diverged and is flanked by a small block of the 48 base-pair satellite, dispersed moderately repeated sequences and a separate short alphoid array. More distal sequences are Y-specific. At the other edge there is much less divergence and the alphoid DNA is flanked by an Alu sequence and the five base-pair satellite.

Improving the care of patients with major trauma in the accident and emergency department.

OBJECTIVE: To determine whether improvement in the care of victims of major trauma could be made by using the revised trauma score as a triage tool to help junior accident and emergency doctors rapidly identify seriously injured patients and thereby call a senior accident and emergency specialist to supervise their resuscitation. DESIGN: Comparison of results of audit of management of all seriously injured patients before and after these measures were introduced. SETTING: Accident and emergency department in an urban hospital. PATIENTS: All seriously injured patients (injury severity score greater than 15) admitted to the department six months before and one year after introduction of the measures. RESULTS: Management errors were reduced from 58% (21/36) to 30% (16/54) (p less than 0.01). Correct treatment rather than improvement in diagnosis or investigation accounted for almost all the improvement. CONCLUSIONS: The management of seriously injured patients in the accident and emergency department can be improved by introducing two simple measures: using the revised trauma score as a triage tool to help junior doctors in the accident and emergency department rapidly identify seriously injured patients, and calling a senior accident and emergency specialist to supervise the resuscitation of all seriously injured patients. IMPLICATIONS: Care of patients in accident and emergency departments can be improved considerably at no additional expense by introducing two simple measures.

Active transport of salicylate by rat jejunum.

Using an improved segmented flow technique the uptake of salicylate in low concentration (1-3 mM) by rat jejunum is found to be steady for ca. 30 min, and is proportional to lumen concentration. At higher lumen concentrations (6-8 mM) the rate of uptake falls from the beginning to very low levels. At low lumen concentrations the tissue fluid concentration rises to approximately three times the lumen concentration without significant effect on the uptake rate. This rise is an exponential function of the net water transport across the intestinal wall. The rate constant is of the same order of magnitude as those found earlier for equilibration of tissue fluid with glucose and wash-out of protein from the tissue fluid. The uptake of salicylate is inhibited irreversibly by 2,4-dinitrophenol (2-4 x 10(-5) M).

Active intestinal transport of methyl red isomers.

Transport across the wall of rat jejunum of two isomers of methyl red has been studied. These isomers, meta-methyl red (MMR) and para-methyl red (PMR), are absorbed against a concentration gradient. Uptake consists of three components: rapid adsorption on to the mucosa, a steady uptake proportional to lumen concentration and a reflux which increases exponentially to a limiting value. A substantial part of the uptake is stored in the mucosa, and some of it is metabolized to colourless derivatives. If methyl red is washed out of the lumen after the mucosa has been loaded, there is some reflux into the lumen, but most of the stored methyl red passses into the secretion. The rate of this transport on to the serosal surface is not noticeably diminished until the adverse concentration gradient exceeds 13-14:1. Once the methyl red has been washed out of the lumen there is no further metabolism of methyl red to colourless derivatives. Shifting the lumen pH from 7.4 to 6.4 whilst keeping the tissue fluid pH constant increases the rate of uptake from the lumen by a factor of only 1.68 although the concentration of unionized methyl red is increased approximately 10-fold. It seems that at pH 7.4 about 90% of the uptake is of the ionized form, and that at pH 6.4 this percentage falls to 50. It is concluded that the methyl reds form fresh examples of foreign organic compounds which can be transported actively by the small intestine and that they can be taken up from the lumen into the mucosa in both ionized and unionized states.

Dependence of intestinal glucose absorption on sodium, studied with a new arterial infusion technique.

1. A new preparation of isolated rat jejunum plus ileum (ca. 100 cm) is described in which a saline infusate is pumped into the superior mesenteric artery, the superior mesenteric vein having been ligated.2. The arterial infusate washes out the tissue spaces: the lumen is perfused in a single pass with a segmented flow as by Fisher & Gardner (1974).3. At an arterial infusion rate of 3 ml./min, steady states are set up in the tissue fluid within 10-15 min: the compositions of the fluids bathing both sides of the mucosa can therefore be controlled.4. The rate of glucose absorption from the lumen falls only gradually when the luminal sodium is replaced by choline abruptly while the tissue fluid sodium is maintained at 144 m-equiv/l. by arterial infusion.5. The rate of glucose absorption from the lumen is unaffected by replacement of sodium in the arterial infusate by choline.6. Ouabain (10(-4) M) in an arterial infusate containing sodium 144 m-equiv/l. causes inhibition of glucose and water absorption from the lumen. There is no effect of ouabain when the arterial infusate contains sodium, 0 or 72 m-equiv/l.7. Arterial ouabain does not reverse the effects of depletion of luminal sodium. Simultaneous removal of luminal sodium and application of arterial ouabain causes faster inhibition of glucose absorption than does either treatment alone.8. Glucose absorption is more likely to depend on rate of efflux of sodium from mucosal cell to tissue fluid than on a sodium gradient at the brush border or on intracellular sodium concentration.

A kinetic approach to the study of absorption of solutes by isolated perfused small intestine.

1. A new technique has been developed for making serial measurements of water and solute absorption from the lumen of isolated small intestine.2. The isolated intestine is perfused in a single pass with a segmented flow of slugs of liquid separated by bubbles of oxygen-carbon dioxide mixture. Simultaneous collections are made of effluent from the lumen and of the fluid which is transported across the mucosa. This latter fluid appears to be a fair sample of the tissue fluid.3. Conditions in the lumen can be changed within less than 5 min. The effects of two or more treatments applied to the same segment of intestine can be determined and the time course of a change in luminal conditions.4. The rate of appearance of solutes on the serosal side depends on the rate of water absorption, and changes exponentially towards a steady state. The rate constant is a function of tissue fluid volume.5. In the steady state the concentration of glucose in the tissue fluid is 71 mM when the luminal concentration is 28 mM, and is 45 mM when the luminal concentration is 8.3 mM.6. For solutes such as glucose for which reflux from tissue fluid to lumen is small relative to flux from lumen to tissue fluid, the time of attainment of a steady state in secretion is usually 50-60 min.7. For solutes such as sodium for which the reflux is relatively high, the steady state may be reached in 15-20 min.8. The K(m) for glucose absorption (14-19 mM) is much lower than is found with unsegmented flow perfusion.9. These findings emphasize problems in interpreting results from other types of intestinal preparation.10. The rate of glucose absorption from the lumen falls only gradually when the luminal sodium concentration is reduced abruptly. In contrast the rate of glucose absorption falls suddenly when the luminal glucose concentration is reduced abruptly. This suggests that glucose absorption is not directly dependent on luminal sodium ions.

Accessibility of heart water to pentoses.

1. The raffinose space, the inulin space and the weight loss on compression of perfused rat hearts are indistinguishable in magnitude.2. The inulin space shows systematic variations from day to day and is also affected by the procedure used for removing surplus water from the perfused heart. It is therefore not safe to use a mean figure of extracellular space in permeability experiments.3. Perfusion of hearts with arabinose solutions alters the intracellular water content. These alterations can be accounted for if it is assumed that the arabinose which penetrates the cells is uniformly distributed in the cell water.4. By appropriate choice of pentose concentration and of time of perfusion it can be shown that all the estimated cell water is accessible to pentose.5. It is concluded that no detectable fraction of the extracellular water is inaccessible to inulin and that no detectable fraction of the intracellular water is inaccessible to pentose.

Kinetics of pentose permeation in the perfused rat heart.

1. The fractional permeation of heart cell water by L-arabinose and D-xylose after a standard period of perfusion is less the higher the extracellular pentose concentration.2. At any given pentose concentration the kinetics of permeation are consistent with passive diffusion through the cytoplasm or with transport across the cell membrane by a saturable carrier.3. The concentration dependence of permeation is inconsistent with passive diffusion.4. The kinetics of L-arabinose permeation are consistent with a carrier having V(max) = 2.2 m-mole/l. extracellular water per min and a half-saturation concentration [K] of 0.5 x 10(-4)M. The corresponding figures for D-xylose are V(max) = 1.4, [K] = 1.6 x 10(-4)M.

The effect of insulin on the kinetics of pentose permeation of the rat heart.

1. The kinetics of permeation of pentoses into the rat heart in the presence of insulin are similar in nature to those in the absence of insulin.2. The half-saturation concentration of the carrier system is increased by insulin in a manner which is independent of insulin concentration.3. The V(max) of the carrier system is altered by insulin in a graded fashion: at low insulin concentrations it may be lower than in the absence of insulin.4. It is shown that these effects can be accounted for if insulin combines with the carrier and thereby increases either the rate constant governing the mobility of the carrier in the membrane or the rate constant governing dissociation of the sugar-carrier complex.5. The effects cannot be accounted for by a graded action of insulin on the membrane as distinct from the carrier.

A diurnal rhythm in the absorption of glucose and water by isolated rat small intestine.

1. Glucose and water absorption by isolated small intestine from rats which have had unrestricted access to food is 50-60% higher at night than during the daytime. 2. When the feeding time is restricted to 06.00-09.00 hr G.M.T. glucose and water absorption rates in the period from 3 to 7 hr after withdrawal of food are almost as high as the rates observed at night-time in the animals with unrestricted feeding. 3. These changes in absorption rates appear to be associated with feeding time and not with the pattern of illumination.

Brodifacoum rodenticide ingestion in a patient with shigellosis.

Factitious disorders are characterized by the intentional feigning or induction of signs and/or symptoms in order to assume the sick role. The spectrum of diseases and symptoms simulated is extensive. Although some patients may seek only the gratifications of the sick role, typically patients seek health care for their afflictions. We report the case of a woman with a history of numerous unexplainable illnesses and laboratory findings who had shigellosis. On routine evaluation, a severe prothrombin coagulopathy was discovered and later determined to be caused by brodifacoum, a "superwarfarin" drug found in potent rodenticides. The patient was successfully treated with daily vitamin K. She continued to deny intentional or accidental ingestion but did consent to psychiatric treatment.

The major centromeric array of alphoid satellite DNA on the human Y chromosome is non-palindromic.

We have determined the orientation of the major centromeric alphoid array on the human Y chromosome. A PCR assay was used to analyse the vector-insert junctions of seven YAC clones previously positioned on two independent Y chromosomes. The orientation is the same at all 10 positions measured. This suggests that the alphoid array is a simple unidirectional repeat throughout and that human centromere structure thus differs from the palindromic organisation found in Schizosaccharomyces pombe.