A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG).

Deletion of the long arm of chromosome 20 represents the most common chromosomal abnormality associated with the myeloproliferative disorders (MPDs) and is also found in other myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Previous studies have identified a common deleted region (CDR) spanning approximately 8 Mb. We have now used G-banding, FISH or microsatellite PCR to analyse 113 patients with a 20q deletion associated with a myeloid malignancy. Our results define a new MPD CDR of 2.7 Mb, an MDS/AML CDR of 2.6 Mb and a combined 'myeloid' CDR of 1.7 Mb. We have also constructed the most detailed physical map of this region to date--a bacterial clone map spanning 5 Mb of the chromosome which contains 456 bacterial clones and 202 DNA markers. Fifty-one expressed sequences were localized within this contig of which 37 lie within the MPD CDR and 20 within the MDS/AML CDR. Of the 16 expressed sequences (six genes and 10 unique ESTs) within the 'myeloid' CDR, five were expressed in both normal bone marrow and purified CD34 positive cells. These data identify a set of genes which are both positional and expression candidates for the target gene(s) on 20q.

Image analysis of gunshot residue on entry wounds. II--A statistical estimation of firing range.

A statistical investigation of the relationship between firing range and the amount and distribution of gunshot residue (GSR), used automated image analysis (IA) to quantify GSR deposit resulting from firings into pig skin, from distances ranging between contact and 45 cm. Overall, for a Ruger .22 semi-automatic rifle using CCI solid point, high velocity ammunition, the total area of GSR deposit on the skin sections decreased in a non-linear fashion with firing range. More specifically there were significant differences in the amount of GSR deposited from shots fired at contact compared with shots fired from distances between 2.5 and 45 cm; and between shots fired from a distance of 20 cm or less, with shots fired at a distance of 30 cm or more. In addition, GSR particles were heavily concentrated in the wound tract only for contact and close range shots at 2.5 cm, while the particle distribution was more uniform between the wound tract and the skin surfaces for shots fired from distances greater than 2.5 cm. Consequently, for future scientific investigations of gunshot fatalities, once standards have been established for the weapon and ammunition type in question, image analysis quantification of GSR deposited in and around the gunshot wound may be capable of providing a reliable, statistical basis for estimating firing range.

Image analysis of gunshot residue on entry wounds. I--The technique and preliminary study.

An automated image analysis (IA) technique has been developed to obtain a measure of the amount (i.e. number and area) of gunshot residue (GSR) particles within and around a gunshot wound. Sample preparation and IA procedures were standardised to improve the reproducibility of the IA measurements of GSR. Measurements of GSR from test firings into goat hide were enhanced by staining the barium and lead components present on the skin sections with Alizarin Red S. The amount of GSR detected on the stained skin sections was compared with backscatter electron micrographs of the same sections. The differences were deemed to be insignificant when the variability in the repeated test firings were taken into consideration. Preliminary results indicated that the decreasing relationship between firing range and the amount of GSR deposited was non-linear, and that for firing ranges of up to 20 cm the amount of GSR deposited from repeated shots fired from the same distance was highly variable.

Age and temperature related changes to the ultrastructure and composition of human bone mineral.

This X-ray diffraction (XRD) investigation of heat-treated human femoral bone showed that the main mineral phase of both unheated bone and bone heated to 600 degrees C resembled that of a poorly crystalline form of hydroxyapatite. The rod-shaped apatite crystals in unheated bone persisted in bone heated up to 400 degrees C. Recrystallization at approximately 600 degrees C, produced larger crystals, which either retained their original morphology or changed to tabular or equidimensional shapes. The size of the apatite crystals in unheated and heated bone specimens was dependent on both temperature and age. When heated above 600 degrees C the crystallinity of the bone mineral increased, and the XRD pattern more closely resembled that of hydroxyapatite. Partial decomposition of the hydroxyapatite phase to calcium oxide above 1000 degrees C, and beta-tricalcium phosphate, alpha-tricalcium phosphate, and calcium oxide phosphate between 1200 degrees C and 1400 degrees C, indicated that the original apatite phase was both calcium deficient and contained carbonate. The relative peak intensities of the thermal decomposition products were related to some extent to the age of the deceased person and reflected the compositional changes that occur during bone aging. Because the thermally induced changes to the composition and ultrastructure of bone mineral were influenced by the age of the individual, this investigation proposed that the heat treatment of bone tissue may offer an alternative way of studying bone aging.

Scanning electron microscope observations of incinerated human femoral bone: a case study.

Fragments of the incinerated remains of a fire victim were studied using scanning electron microscopy and microradiography. These observations were then compared with the heat-induced alterations found in laboratory heat-treated human bone. The incinerated bone fragments exhibited a range of colours, including black, grey and white, concomitant with alterations to the ultrastructure and microstructure of the bone tissue. The colour of the incinerated bone tissue, and the crystal habit and size associated with each region of colour, indicated a gradual decrease in the temperature attained in the bone, as a function of the radial distance from the outer cortical bone surface. A maximum temperature in the range approximately 1000-1200 degrees C had been attained in the outer cortical bone regions that were white in colour. A minimum temperature of 300 degrees C had been attained in the inner cortical bone regions that were black in colour. The period of time over which the fire attained the maximum temperature was inferred to be too short for the bone tissue to have reached an equilibrium with the surrounding environment, as the fire was due to a sudden ignition. However, the minimum temperature recorded was attained for a longer period of time as the organic contents of the Haversian canals and the medullary cavity had been completely combusted. From examinations of the spherical-type crystal size in the grey regions of cortical bone, the habit of the hexagonal-type crystals in the white regions of bone, and the preferred orientation of the mineralised collagen fibrils in the Haversian canals situated in the black regions of cortical bone, it was suggested that the deceased person was a young-to-mature adult, possibly aged 20-40 years.

Scanning electron microscope observations of heat-treated human bone.

This report describes the heat-induced alterations in human bone tissue observed using scanning electron microscopy and microradiography. Femoral bone samples were taken from persons varying in age from 1 year to 97 years at the time of their death. The bone was heated at selected temperatures in the range 200-1600 degrees C for periods of 2, 12, 18 and 24 h. Macroscopically, changes in colour occurred, together with some shrinkage, fracturing and distortion. However, dramatic changes occurred at the ultrastructural level. These changes included the progressive combustion of the organic portion of the bone tissue up to 400 degrees C and recrystallisation of the bone mineral beginning at 600 degrees C. Recrystallisation produced a range of crystal morphologies: spherical, hexagonal, platelets, rosettes and irregular. Crystal growth occurred at temperatures > 600 degrees C. Sintering led to fusion of crystals at 1000 degrees C. This process continued up to temperatures > 1400 degrees C. At 1600 degrees C the bone mineral melted. On heating, the morphology of crystals formed, and the ultrastructural changes which occurred, were found to be related to the age of the deceased, the temperature to which the bone had been heated and the duration of heating. These results are of importance to forensic scientists, arson investigators and paleoarcheologists in their investigation of cremated human bones, particularly when only fragments of bone are available, in order to determine something of the life history of the deceased and the circumstances surrounding the death.

The distribution of brominated long-chain fatty acids in sponge and symbiont cell types from the tropical marine sponge Amphimedon terpenensis.

The tropical marine sponge Amphimedon terpenensis (family Niphatidae, order Haplosclerida) has previously been shown to possess unusual lipids, including unusual fatty acids. The biosynthetic origin of these fatty acids is of interest as the sponge supports a significant population of eubacterial and cyanobacterial symbionts. The total fatty acid composition of the sponge was analyzed by gas chromatography/mass spectrometry of the methyl esters. Among the most abundant of the fatty acids in intact tissue were 16:0, 18:0 and 3,7,11,15-tetramethyl-hexadecanoic (phytanic) acid. In addition, three brominated fatty acids, (5E,9Z)-6-bromo-5,9-tetracosadienoic acid (24:2Br), (5E,9Z)-6-bromo-5,9-pentacosadienoic acid (25:2Br) and (5E,9Z)-6-bromo-5,9-hexacosadienoic acid (26:2Br) were also present. The three brominated fatty acids, together with phytanic acid, were isolated from both ectosomal (superficial) and choanosomal (internal) regions of the sponge. Analysis of extracts prepared from sponge/symbiont cells, partitioned by density gradient centrifugation on Ficoll, indicated that phytanic acid and the three brominated fatty acids were associated with sponge cells only. Further, a fatty acid methyl ester sample from intact tissue of A. terpenensis was partitioned according to phospholipid class, and the brominated fatty acids were shown to be associated with the phosphatidylserine and phosphatidylethanolamine fractions that are commonly present in marine sponge lipids. The phosphatidylcholine and phosphatidylglycerol fractions were rich in the relatively shorter chain fatty acids (16:0 and 18:0). The association of brominated long-chain fatty acids (LCFA) with sponge cells has been confirmed. The findings allow comment on the use of fatty acid profiles in chemotaxonomy and permit further interpretation of LCFA biosynthetic pathways in sponges.(ABSTRACT TRUNCATED AT 250 WORDS)

Rsr1 and Rap1 GTPases are activated by the same GTPase-activating protein and require threonine 65 for their activation.

The Rsr1 protein of Saccharomyces cerevisiae has been shown to be essential for bud site selection (Bender, A., and Pringle, J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9976-9980). This protein of 272 amino acids shares approximately 50% sequence identity with both Ras and Rap GTPases. However, neither GTP binding nor GTPase activity of the Rsr1 protein has been reported. The Rsr1 protein shares with human Rap1 GTPases the four specific motifs, i.e. Gly-12, residues 32-40, Ala-59, and residues 64-70, that are required for GAP3-dependent activation of the Rap1 GTPases. In this paper we demonstrate that the intrinsic GTPase activity of the Rsr1 protein is stimulated by GAP3 purified from bovine brain cytosol. The Rsr1 GTPase is not activated by either GAP1 or GAP2 which are specific for the Ras and Rho GTPases, respectively. Thus, it appears that the Rsr1 GTPase is a new member of the Rap1 GTPase family. Replacement of Gly-12 by Val in the Rsr1 GTPase completely abolishes the GAP3-dependent activation. The chimeric GTPases, Ras(1-60)/Rsr1(61-168) and Rsr1(1-65)/Ras(66-189), are activated by GAP3 but not by GAP1. Replacement of Thr-65 by Ser in the latter chimeric GTPase completely abolishes the GAP3-dependent activation, indicating that Thr-65 is required for distinguishing GAP3 from GAP1. We have previously shown that Gln-61 and Ser-65 are sufficient to determine the GAP1 specificity. Replacement of Thr-35 by Ala in the common effector domain (residues 32-40) of the chimeric Ras/Rsr1 GTPases completely abolishes GAP3-dependent activation.

Metabolism of 4-hydroxyanisole: identification of major urinary excretory products.

The human metabolism of 4-hydroxyanisole was investigated by the analysis of urine samples from melanoma patients treated with this substance. The samples were hydrolyzed with glucoronidase and/or arylsulphatase, extracted with ethyl acetate, and, after derivatization with pentafluoropropionylanhydride, analyzed by gas chromatography-mass spectrometry. We were able to identify peaks by their retention times and mass spectra corresponding to 4-hydroxyanisole, 3,4-dihydroxyanisole, and two of its o-methyl derivatives, namely, 3-hydroxy-4-methoxy- and 4-hydroxy-3-methoxyanisole. We also detected a peak of hydroquinone, which may have originated (at least partly) from 4-hydroxyanisole. All the above-mentioned compounds were excreted predominantly as sulphates and glucuronides. Only a small proportion of the substances was present in urine in an unconjugated form. Our results demonstrate that 3,4-dihydroxyanisole is the most important metabolite of 4-hydroxyanisole.

Major primary cytotoxic product of 4-hydroxyanisole oxidation by mushroom tyrosinase is 4-methoxy ortho benzoquinone.

It has been shown previously that the initial product of mushroom tyrosinase-catalysed oxidation of the monophenol 4-hydroxyanisole (4HA) is 4-methoxy ortho benzoquinone (4-MOB). This study presents evidence that 4-MOB is primarily responsible for the cytotoxicity of 4HA oxidation products in vitro. Equivalent toxicity in a model system was produced by products of tyrosinase catalysed oxidation of 4HA and by synthetic 4-MOB. Cytotoxicity was estimated both by a blebbing assay and by plating efficiency of exposed cells. HPLC analysis of the reaction mixture revealed a positive correlation between cytotoxicity and 4-MOB concentration.

Delay in entry into S phase after heat shock.

Synchronized cells of the Harding Passey melanoma grown in culture were given a heat shock treatment of 44 degrees C for 36 min. Thymidine incorporation was measured at frequent intervals after heat shock to determine the time of onset of the next DNA synthetic period. If the heat shock was given at the end of G1, the following S was delayed by 20 hr. Heating at other times in the cell cycle resulted in an even longer interval before the onset of S. The end of G1 was also the most resistant to hyperthermic killing and to the effect of heat on the magnitude of thymidine incorporation in the following S. Heating the cells a second time did not repeat the effect of the first treatment unless the second heat shock treatment was at a considerably higher temperature. Thus thermotolerance to heat shock killing also applies to cell-cycle delay.

Barriers to informed consent.

The law of informed consent seeks to actively involve patients in decision making. Most authorities agree that this involvement has not occurred but disagree about why. Some suggest that patients are incapable of understanding medical issues and others that physicians have not explained issues clearly or extensively enough. We observed decision making in several hospital settings and found other significant barriers to patient participation. These barriers include the fact that treatment decisions take place over a long period; there are often many decisions to be made; although patients want information about treatment, they typically believe that decision making is the physician's task; physicians do not understand the rationale for the patient's role in decisions; and the medical decision-making process often involves so many people that the patient does not know who is responsible.

Pancreatic abscess following acute pancreatitis.

Pancreatic abscess is probably the most serious complication of acute pancreatitis. During the ten-year period from 1966 to 1975, twenty-eight patients with pancreatic abscess following acute pancreatitis were treated by surgical drainage. A review of these cases revealed that there was a lull in the clinical course of the antecedent pancreatitis prior to the time of surgical drainage in 70% of the cases. Despite an aggressive surgical approach, there were major postoperative problems in 26 patients. Sepsis persisted in 14 patients. Major gastrointestinal hemorrhage occurred in seven, intra-abdominal bleeding in nine, and fistulization in 13. Fourteen patients died (a mortality of 50%). The operative treatment of pancreatic abscess must be aggressive and persistent. In addition to extensive drainage with soft sump drains, vigilance must be exercised to avoid pressure against bowel or major vessels. Reoperation should be considered if postoperative improvement is not sustained.