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A multicentre study involving 36 out of 38 German medico-legal Institutes discovered 784 non-natural fatalities among 78,000 external examinations before cremation (1%) in 1995. These included 169 accidents, 16 suicides, 3 homicides and 589 deaths in connection with medical treatment. In the remaining 7 cases, a belated differentiation between homicide and accident was not successful. A linear projection results in 1980 non-natural fatalities among all 197,000 external examinations before cremation. This number includes 8 homicides but due to considerable inhomogeneities, a number of 40 homicides appears to be realistic. Consequently, abolishing the mandatory external examination before cremation would be equivalent to an official renouncement of solving relevant non-natural deaths. In case of a thorough external examination, a total of 18,000 non-natural fatalities including approximately 180 homicides could be detected per year during the first external examinations (n = 885,000).

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.

In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 microM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10 microM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1 microgram of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (approximately 5 microM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 microM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 x 10(6) theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.

On-line affinity capillary electrophoresis-electrospray ionization-mass spectrometry (ACE-MS) was used for the simultaneous measurement of multiple binding constants of an all-D-tetrapeptide library to the model receptor, vancomycin. Determination of Kd values for the 19 peptides of the form Fmoc-DXYA is demonstrated. The data are compared with the results obtained for individual compounds using ACE-UV, and good correlation between the two detection methods is shown. Simultaneous determination of multiple Kd values by ACE-MS is achieved in one set of experiments, whereas only one Kd value can be obtained by ACE-UV during the same time. ACE-MS measures multiple binding constants in solution in a fast and reliable manner using femtomole amounts of samples.

When a bullet strikes an interposed object before entering a body, the bullet may fragment, or particles from the interposed object may be accelerated. Such secondary missiles may cause multiple entrance wounds if a body is located in line with their flight path. In the unusual case reported here, a silencer acted as an interposed object. The bullet disintegrated completely after impact with the central part of the silencer baffles, which were misaligned. Some bullet fragments were retained inside the silencer, and some exited as a collection of missiles, comparable to shotgun pellets. From a distance of several meters, the fragments wounded the victim at seven different anatomic sites. Use of a silencer should be considered when bullet fragments are recovered from a scene or victim but no other appropriate interposed object is found.

In a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long read-lengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al., Anal. Chem. 1996, 68, 3305-3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa, with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measurements to characterize the polymer, good batch-to-batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solutions containing 2% w/w of LPA made by emulsion polymerization were simple to prepare, resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied, allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read-length of more than 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.

The differentiation between contact bloodstains and stains produced by projected droplets on fabric can be crucial in crime scene reconstruction since suspects can explain bloodstains on their clothing by contact with the victim post mortem. Experimental smear and contact pressure stains on three different types of fabric were compared with stains produced by falling and projected droplets ("dynamic" stains) of equivalent size. The morphology of the small stains (0.1-10 microliters) was investigated with a macroscope. Characteristic for "dynamic" stains are symmetry and rhythmicity (secondary droplets) of the stain and a zonal drying pattern. Contact stains lack the characteristic features of "dynamic" stains and show a clear impregnation of the material resulting in a blood-soaked reverse side of cambric and cotton and in a paler overall colour. The mode of formation of microstains (blood volume 1 < microliter) on irregular surfaces (e.g. terry cloth) can be difficult to determine since the rough surface structure and the small blood volume reduce the characteristics of dynamic stains. In these cases, comparison with experimental stains on the same surface material is recommended.

The tissue disruption inside the brain after experimental gunshots to the head was investigated with special reference to secondary bone missiles and intracranial pressure effects such as cortical contusion and deep intracerebral haemorrhages. The evidential value of various examination methods is compared. 9 mm Parabellum ammunition was fired to the temporal region of calves (n = 10) from a distance of 0-10 cm. Plain film radiography, CT, MRI, visual inspection and histology were performed on every brain. The tissue disruption of the permanent tract is delineated best by artefact-free MRI. Cortical contusions and deep intracerebral haemorrhages were detected infrequently by visual inspection and imaging techniques although they were present in every brain as verified by histology. These injuries remote from the tract increase cerebral wounding compared to non-confined tissue. In particular, the brain stem and central areas were frequent sites of haemorrhages, which can be expected to have serious and immediate consequences. Ectopic bone fragments were found in all brains using CT scans. Bone fragments were located inside clearly enlarged permanent tracts or were driven into brain tissue. In the latter cases, secondary shot channels up to 4 cm in length could be verified by histology. Cortical contusions and intracerebral haemorrhages can only be detected reliably by histology. The localization of bone fragments requires CT scans. Therefore, a detailed examination is accomplished best by a combination of the methods applied in this study.

Lethal injuries of the thorax due to shots fired from blank cartridges calibre 8 mm are reported in three cases. The muzzle of the weapon was in contact with the left side of the breast (contact discharge) and injuries to bones were absent in all three cases. In two of the cases the pericardium was not involved but the anterior wall of the right heart ventricle was ruptured and death was due to cardiac tamponade. In the third case the pericardial sac and the left ventricle were both ruptured and the victim died due to rapid exsanguination. The cases demonstrate that the gas pressure from the exploding propellent of blank ammunition can be powerful enough to penetrate the thoracic wall.

In clinical medicine, severe keto- or lactic acidosis associated with vomiting, nausea, abdominal pain, tachycardia or pathological respiration, has been described in chronic alcoholics. This study reports on fatalities of chronic alcoholics where the cause of death could not be determined by thorough autopsy, histology and toxicology including determination of alcohol concentration. In a first series, acetone was determined in the blood of such chronic alcoholics (n = 24), diabetics with metabolic decompensation (n = 7), cases of hypothermia (n = 7) and controls (n = 218). Among the 24 chronic alcoholics where the cause of death was unknown, 9 cases showed very high levels of acetone (74-400 mg/l). These comprised 6 cases without additional findings and 3 cases where a second patho-mechanism such as intoxication possibly contributed to the cause of death. In a second series, the sum values according to Traub (lactate/glucose) were determined in cerebrospinal liquor of chronic alcoholics with undetermined cause of death (n = 45), diabetics (n = 6) and controls (n = 39). Among the 45 alcoholics, 17 cases showed very high sum values (294-594 mg/dl) including 8 cases where non-lethal intoxications may have contributed to the final outcome. Other causes of a ketoacidosis or lactic acidosis (e.g. diabetes) were excluded in both groups of alcoholics. Consequently, ketoacidosis and lactic acidosis can be the cause of death of chronic alcoholics in a considerable number of cases where no pathomorphological or toxicological changes are present. A scheme for medical and laboratory examination is described.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), has been used for structure characterization of 1-aminopyrene-3,6,8-trisulfonate (APTS)-derivatized oligosaccharides previously separated by capillary electrophoresis (CE). The resolved components were first isolated by employing an automated high-resolution fraction collector. Using on-probe sample cleanup with a cation-exchange resin and a matrix mixture of (1:1) 6-hydroxypicolinic acid and 3-hydroxypicolinic acid, APTS-labeled oligosaccharides were successfully detected by MALDI-TOF MS in the negative ionization mode. Each APTS-labeled oligosaccharide produced single [M-H]-1 peaks. Detection limits for standard APTS-derivatized oligosaccharide (maltoheptaose) were down to 30 fmol. APTS labeled maltooligosaccharides and various standard carbohydrates were separated and collected by CE, followed by molecular weight determination with MALDI-TOF MS. The mass spectral characterization enhances the power of the CE analysis of oligosaccharides.

Using beta-endorphin as a model system, a new microscale solution-based approach for linear epitope mapping based on affinity capillary electrophoresis-mass spectrometry (ACE-MS) is demonstrated. Tryptic peptides are separated in a neutral coated capillary and monitored by ultraviolet absorbance and electrospray mass spectrometry. Then, following injection of the peptide digest mixture, anti-beta-endorphin antibody is injected. The peptide, which binds to the antibody, is captured and disappears from its migration time. Following this subtraction-screening procedure, the binding of the individually synthesized or isolated immunoreactive peptide is examined by the ACE-MS procedure to confirm that the epitope resides on the peptide. A series of truncated peptides can then be made and the precise epitope determined by ACE-MS. The method utilizes low femtomole amounts of antibody and peptide digest per run and is rapid and easily automatable.

A case of physical activity following a suicidal gunshot to the head including perforation of the left frontal lobe from a .22 rimfire rifle is reported. Subsequently, the man was able to walk a distance of more than 100 m and to reload manually. The special wound ballistic features of the head leading to increased intracranial projectile effects are discussed and the morphological changes such as cortical contusion zones, intracerebral hemorrhages or indirect skull fractures are explained. The analysis of 38 cases of head shots followed by physical activity reveals that injury to certain CNS-areas or the use of centre fire rifles and large handguns usually excludes a potential for subsequent physical activity. But physical activity is possible if the trajectory is restricted to the frontal brain or one temporal lobe only and if a projectile of low wounding potential has been used for this gunshot.

Microfabricated multiple-channel glass chips were successfully interfaced to an electrospray ionization mass spectrometer (ESI-MS). The microchip device was fabricated by standard photolithographic, wet chemical etching, and thermal bonding procedures. A high voltage was applied individually from each buffer reservoir for spraying sample sequentially from each channel. With the sampling orifice of the MS grounded, it was found that a liquid flow of 100-200 nL/min was necessary to maintain a stable electrospray. The detection limit of the microchip MS experiment for myoglobin was found to be lower than 6 x 10(-8) M. Samples in 75% methanol were successfully analyzed with good sensitivity, as were aqueous samples. The parallel mutliple-channel microchip system allowed ESI-MS analysis of different samples of standard peptides and proteins in one chip.

In continuation of our work to develop an integrated multichannel microchip interfaced to electrospray mass spectrometry (ESI-MS), this paper demonstrates one of several applications of this approach in monitoring tryptic digestion products. The multichannel microchip allowed integration of sample preparation onto the microchip to facilitate the analysis process. Melittin was selected as a model oligopeptide because it possesses a cluster of four adjacent basic residues which enable probing the site specificity of trypsin as a function of digest times. Reactions were performed on-chip in different wells for specific time periods and then analyzed by infusion from the microchip by ESI-MS, using leucine enkephalin as internal standard. The rate of formation and disappearance of the molecular ion and individual fragments was followed for a melittin to trypsin concentration ratio of 300:1. The results indicate the potential of integrating enzymatic reactions with multichannel microchip ESI-MS for automated optimization of reaction condition while consuming only small amounts of sample.

Out of 138 clearly defined gunshot suicides which were autopsied, 11 persons (8%) fired two or more gunshots to the body. From these 11, 5 cases involved 2 gunshots to the head where the bullets fired first had missed the brain. The trajectories were restricted to the chest in three cases and a combination of gunshots to the head and chest including two perforating heart wounds without immediate incapacitation occurred in three more cases. Reliable incapacitation is based on physiological effects (tissue disruption) and can only be achieved by decreasing the functioning capability of the CNS. This can be accomplished by direct disruption of brain tissue or indirectly by cerebral hypoxemia from massive bleeding. Targets of immediate incapacitation are restricted to certain CNS areas and targets of rapid incapacitation include the heart, the (thoracic) aorta and the pulmonary artery. Other major blood vessels and major organs (lungs, kidneys, liver, spleen) constitute targets of delayed incapacitation. This general classification can be derived from the literature and is illustrated by the cases presented. A thorough post mortem can exclude or quantify the potential for physical activity. Typical features of single gunshot suicides such as contact shots, classical entrance wound sites and soot/backspatter on a hand also occur in multiple gunshot suicides.

Backspatter is the ejection of biological material from a gunshot entrance wound against the line of fire. Backspatter of blood was investigated experimentally in transverse gunshots to the heads of calves (n = 9) from shooting distances of 0-10 cm. The resulting bloodstains were documented on white paper placed horizontally 60 cm below the impact site. The morphology of bloodstains and the distribution of microstains (diameter < 0.5 mm) is reported. The number of microbackspatter stains per gunshot varied between 39 and 262 and the maximum travelling distance was 69 cm while the vast majority of micro-droplets accumulated between 0 and 40 cm. The direction a single droplet can take comprises every possible angle between the most tangential ones to the skin surface. Microstains exclusively were circular to slightly oval. The morphology of macrobackspatter stains (diameter > 0.5 mm) varied from round to elongated with circular, drop-like and stains in the form of exclamation marks predominating. Small macrostains (0.5-4 mm) made up more than 90% of the macrostains and no systematic relationship between distance travelled and size of the stains could be established. The necessity of appropriate lighting and magnification in the investigation of surfaces for backspatter is stressed because many microstains are located in the proximity of the entrance wound where the firearm and the shooting hand are located in cases of close-range shots.

Three immunohistochemically different myelomonocytic subtypes, i.e. MRP8, MRP14 and 27E10 were quantitatively evaluated in the intraalveolar, alveolar-interstitial and alveolar-intracapillary lung compartments. Lung sections from 5 major groups with defined causes of death, i.e. drowning and death during immersion (DI), cerebral/intracranial haemorrhages (CH), sudden cardiac deaths (SCD), hanging and throttling (HT) and immediate trauma deaths (ITD) were stained and the positive cells counted. The results show clear differences of the cell numbers on average. Among the different compartments the intracapillary cell count exhibits the highest numbers. If the cell counts are compared to the different causes of death, DI shows the highest values and ITD the lowest. The individual values, however, show considerable variations in all compartments and especially in the low cell count range. Within the DI group two subgroups can be differentiated, one having low and the other one having high cell numbers. This can be due to the type of agony, i.e. drowning versus immersion/hydrocution, or to resuscitation attempts or to a combination of both factors.

A rugged, stable liquid sheath interfacing procedure for on-line CE/MS is described. This procedure combines optimized component sizes, tapered capillary tips, and adjustment of the capillary with respect to the liquid sheath tube during electrospray operation (active capillary positioning) to establish a stable electrospray quickly and reliably. The interface is especially effective at low flow rates, and CE/MS with a liquid sheath flow rate of 250 nL/min has been achieved. Active capillary positioning also allows on-line capillary isoelectric focusing mass spectrometry with in-probe focusing. In this application, the capillary is retreated into the liquid sheath tube, which becomes a microreservoir. After focusing, the capillary is returned to its proper position for electrospray, and focused zones are mobilized into the mass spectrometer for on-line detection. Both active capillary positioning and in-probe focusing readily lend themselves to automation.

High speed capillary electrophoresis-laser-induced fluorescence (CE-LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA-ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Bluescript II phagemid. LCR-CE-LIF was then applied to detect point mutations in human mitochondrial DNA, resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatable.

A miniaturized, integrated capillary electrophoresis-ultraviolet detection-electrospray ionization-mass spectrometry (CE-UV-ESI-MS) interface has been constructed and evaluated. The device incorporates a fiber optic detection cell close to the electrospray tip to allow UV monitoring of separated zones just prior to their admittance into the mass spectrometer. This configuration provides precise information about the time when UV-active zones enter the electrospray and allows easy location of analyte mass information in the ion current profile. The miniaturized dimensions of the interface allow the use of short capillaries for fast separations.

Capillary electrophoresis with a replaceable linear polyacrylamide matrix operated at elevated column temperatures of 55 degrees and 60 degrees C was used to extend the separation of DNA sequencing fragments to lengths greater than 800 bases. A solid-state heater was employed to provide stable, uniform temperature control over a significant portion of the capillary. The polymer matrix, 3% w/v linear polyacrylamide in a denaturing buffer, was replaced in the capillary after each run. Using dye-labeled primers and Sequenase chemistry on an M13mp18 single-stranded template, four-color separations for the sequencing products were obtained, with read lengths in excess of 800 bases. This paper also briefly discusses the effects of buffer denaturants and capillary temperature on separation speed, resolution, and gel compression.

A procedure is presented for the separation of underivatized oligosaccharides by capillary electrophoresis (CE) with a phytic acid-borate buffer system. The presence of the phytic acid ion-pairing agent greatly increases resolution between oligosaccharides in the complex mixtures studied, which was demonstrated by the separation of oligosaccharides originating from various immunoglobulin G antibodies and CTLA4Ig, a biologic fusion protein. The conditions also resolve neutral oligosaccharides, usually a major CE limitation. High-performance anion-exchange chromatography with pulsed amperometric detection, a standard technique for oligosaccharide and sugar analysis, is used as a reference method to analyze some of the complex oligosaccharide mixtures.

The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.

Two cases illustrating the difficult differential diagnosis of left-sided pain in the coincidence of nephropathy and aortic aneurysm are described. In both cases an aortic aneurysm was discovered, although the aneurysm caused the pain in the side in only one case. In particular, older men with recently occurring side or back pain must be examined with regard to aortic aneurysm. As the classic symptoms of a ruptured aneurysm are frequently disguised, aneurysm must always be considered as a cause of left-sided pain.

Two strategies for DNA sequencing by primer walking using short oligonucleotide primer libraries have been successfully employed along with capillary electrophoresis using replaceable polymer solutions of linear polyacrylamide and fluorescence detection. A 3.5-kb stretch of the single-stranded M13mp18 template was sequenced with T7 PRISM dye-terminator/Sequenase chemistry. An in-house base-calling program offered read lengths of roughly 450 bases with an average of 97.8% accuracy.

A quantitative competitive polymerase chain reaction (PCR) assay for the detection and quantification of HIV-1 has been developed using capillary electrophoresis. Separation of the PCR products is carried out in coated capillaries filled with replaceable linear polyacrylamide, and detection is performed using laser-induced fluorescence. The quantitative capabilities of this assay are described. We show results from analysis of a noninfectious plasmid encoding the HIV genome and integrated proviral DNA. Potential applications of this assay are discussed.

Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity.

Criterias for the differentiation between hunting accidents and suicides are explained and discussed. A thorough investigation of this kind should always include autopsy, examination of the firearm, determination of the shooting distance and examination of the scene including an effort to reconstruct the events. Typical findings in suicides are contact shots, entrance wounds of the head, chest or upper abdomen, downward trajectories and evidence (gunshot residue, backspatter) suggesting a hand had been used to steady the muzzle. Typical findings in hunting accidents are entrance wounds of the extremities, the lower abdomen or the back, worn out or defective safety devices, upward trajectories and trajectories that are incompatible with possible and intended positions of the firearm. Each case has to be examined individually and the findings should be interpreted as an entity.

The protein product of the mouse preimplantation embryo development (Ped) gene, which controls the rate of preimplantation embryonic cleavage division and subsequent embryo survival, is the Qa-2 antigen. This major histocompatibility complex (MHC) class I b protein is encoded by four genes, Q6, Q7, Q8, and Q9. The present study was undertaken to begin to elucidate which of the four Qa-2-encoding genes are responsible for the Ped gene phenotype in the C57BL/6 mouse (H2(b)). First, restriction maps of the four genes, using 25 restriction enzymes, were created. The RE maps confirmed that Q6 is similar to Q8 and Q7 is similar to Q9, but that the Q6/Q8 gene pair differs from the Q7/Q9 gene pair. The genomic DNA sequences of Q6 and Q8 were determined, as well as the DNA sequences of exons 4 - 8 of Q9, and the 5' regulatory regions of Q6, Q8, and Q9. This DNA sequence information, combined with the published DNA sequence information for the entire Q7 gene and exons 1 - 3 of Q9, allowed us to design primers for reverse transcription-polymerase chain reaction that could distinguish which of the four genes were transcribed in mouse lymphocytes and embryos. It was found that all four genes are transcribed in lymphocytes, but only Q7 and Q9 are transcribed in mouse embryos. Thus, both Q7 and Q9 are candidates for the genes responsible for the Ped gene phenotype.

Backspatter is the ejection of biological material from a gunshot entrance wound against the line of fire. This phenomenon was investigated experimentally in transverse gunshots to the heads of calves (n = 9) using two types of 9 mm Parabellum ammunition from shooting distances of 0-10 cm. The resulting bloodstains were documented on white paper placed horizontally 60 cm below the impact site. In this report the analysis was restricted to stains with a diameter > 0.5 mm. Backspatter was documented after every gunshot. The number of stains varied from 31-324 per gunshot and appeared to be independent of the shooting distance. The maximum distance droplets travelled varied from 72-119 cm. The majority of droplets accumulated between 0 and 50 cm. The number of droplets and the distances travelled should be higher in man for anatomical reasons. The direction a single droplet can take comprises every possible angle between the most tangential ones to the skin surface. This resulted in a semi-circle of 180 degrees covered with stains. Skin ruptures of the entrance wound were not observed. The succession of events was documented on high speed film and started with the recoil of the firearm, immediately followed by a blow-out effect of the skin. Large droplets exited approximately 0.7-4 ms after the bullet impacted the skin. The calculated minimum initial velocity of these droplets was 13-61 m/s. Backspatter from gunshots to the head likely is caused by the hot gases expanding subcutaneously and by cavitation-related intracranial overpressure and tail splashing. In three out of nine gunshots, secondary backspatter additionally occurred as a result of droplets produced by a stream of blood from the entrance wound impacting the paper surface.

Accidental hunting firearm injuries and fatalities (257 cases from 1961 to 1992) were evaluated in detail. Most persons responsible for the accident were more than 40 years old and experienced in hunting, and 26% of the gunshot wounds were fatal. In 77% of cases the victim was shot by another person and in 23% the wound was self-inflicted. The firearms/ammunition used were pellets from shotguns (63%), bullets from rifles (31%), shotgun slugs (3.5%) and bullets from handguns (2.5%). In 22% of all accidents from pellets severe eyeball injuries were involved, and 38% of the wounds were caused from a distance of 5 m or less, including all self-inflicted injuries. The most frequent factors responsible for the accident were: improper handling of the firearm (37%), failure to notice the victim (24.1%), covering the victim while swinging on the game (14.8%), ricocheting projectiles (13.6%), inadequate storage of the firearm (11.7%) and mistaking the victim for game (9.3%). In some cases more than one factor contributed to the accident. Defective firearm/ammunition, as the only non-human factor, was involved in only 1.6% of accidents. Some aspects of the prevention and the forensic investigation of hunting accidents are discussed.

In this study, the use of capillary isoelectric focusing (cIEF) as a micropreparative tool for protein analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated. A newly designed, automated, collection interface equipped with a fiber-optic UV detector and a sheath flow connection was employed for collection of protein fractions. Multiple fractions were collected during a single cIEF run and further analyzed by MALDI-TOF-MS for mass assignment. The feasibility of the method was tested with a mixture of model proteins with different isoelectric points and molecular masses, and with variants of human hemoglobins differing in pI, but with negligible difference in M(r). Some practical considerations of the collection procedure and subsequent TOF analysis are presented.

A high-precision fraction collector for capillary electrophoresis has been developed. The device utilizes detection close to the end of the capillary and a sheath liquid at the exit of the capillary, allowing continuous collection (i.e., uninterrupted applied electrical field) of multiple species. The role of the sheath liquid flow rate and position of detection in the column on the collection precision was assessed. Fiber-optic detection at approximately 1 cm before the exit end of the capillary was found effective for precise timing of the collection. Up to 60 fractions of microliter or smaller volumes could be automatically collected into capillaries used as collection vials. The collection capillaries were placed on a cylinder, and a computer-controlled stepping motor aligned the appropriate capillary with the column exit. The effectiveness of the fraction collector was demonstrated in the collection of all 11 fragments of the HaeIII restriction digest of phi X-174 plasmid DNA. Polymerase chain reaction amplification of the 271 and 281 bp fragments revealed an inversion of the size-dependent migration order.

Capillary electrophoresis (CE), using a replaceable polymer matrix and two-dye laser-induced fluorescence has been applied to single strand conformational polymorphism (SSCP). Two-dye laser-induced fluorescence has been used for improved strand identification over a single-dye approach. Conditions suitable in the capillary format for rapid separation and high resolution have been explored. The influence of separation parameters such as temperature and matrix composition on separation in SSCP was first determined. Short analysis times allowed for fast screening of optimal separation conditions of the sample. Based on these results, the two strands of a standard 255 bp fragment of the lacI gene were resolved within 25 min with replaceable linear polyacrylamide as a separation matrix. The method was then applied to the detection of different mutations, in the presence of wild type, of a 276 bp fragment of the insulin-like growth factor 1-binding protein 1 (IGF1-BP3) gene.

Two suicides with home-made guns firing conventional ammunition are reported. Three different classes of homemade guns can be distinguished. Special characteristics of the interior and exterior ballistics of the home-made guns caused unusual muzzle imprints, intensive soot deposits at the entrance wounds and on the hands, intensive CO-effects, burns and in one case a skin laceration of the hand holding the weapon. The bullets showed a reduced penetration depth, characteristic firing marks were missing.

A man committed suicide by a contact shot to the left chest from a nail-gun. The nail caused a slit-like entrance wound and a penetrating heart injury. The front side of the nailgun led to an outer "muzzle imprint", the muzzle and the nail to an inner "muzzle imprint". Wound ballistics of nails are discussed.

Capillary electrophoresis (CE) with a replaceable linear polyacrylamide (LPA) sieving matrix was used to examine sequence-dependent migration of double-stranded DNA fragments. It has been found that DNA conformational effects were significant under high electric field separations, especially using high resolution matrices. Compared to linear DNA-ladder standards, both anomalously slow and rapid DNA fragments were observed, with the degree of anomalous migration depending on the electric field strength, polymer concentration, column temperature, and background electrolyte (denaturants, sodium and magnesium ions, DNA-intercalating dyes). By selecting a combination of electrophoretic conditions (e.g. 3% T LPA, elevated capillary temperature, lower electric field strength and addition of DNA intercalating dyes), molecular weight dependent separations were closely restored.

There are two distinct mechanisms of ballistic injury. Crushing of tissue resulting in a permanent tract is the primary factor in wounding of most tissues and most body regions. Temporary cavitation causes radial tissue displacement and subsequent shearing, compression and especially stretching of tissue analogous to blunt trauma. In contrast to the effect in elastic tissue, temporary cavitation can contribute substantially to wounding of inelastic tissue, such as the brain. This is the case in penetrating gunshot wounds to the head. Additionally, the penetration of the bony cranium can produce secondary missiles in the form of bone or bullet fragments and a tendency of the bullet to deformation and early yaw. Most important, wounding resulting from temporary cavitation is greatly augmented by the confined space provided by the unyielding walls of the skull. Bone contact and enhanced effects of temporary cavitation result in an enlarged zone of disintegrated tissue and in high intracranial peak pressures. Morphological signs of powerful intracranial pressure effects are cortical contusion zones, indirect skull fractures and perivascular haemorrhages remote from the tract. Depending on ballistic and anatomical parameters, the intracranial effect varies from slightly more severe injury than in isolated soft tissue to an "explosive" type of injury with comminuted fractures of the skull and laceration of the brain. Incapacitation is the physiologically based inability to perform complex and longer lasting movements independent of consciousness or intention. Immediate incapacitation is possible following cranio-cerebral gunshot wounds or wounds that disrupt the upper cervical spinal cord only. Rapid incapacitation can be produced by massive bleeding from major vessels or the heart. Immediate incapacitation is the result of primary intracranial effects of the bullet. A mechanism similar to commotion cerebri applied extracranially does not exist in cases of penetrating gunshot wounds to the head.

A man committed suicide with a modified signal pen gun and a .22 lr HV HP bullet. The contact shot to the left chest led to a conventional entrance wound. The projectile from the unconventional weapon caused a penetrating wound and was not deformed instead of the expected perforation and deformation. The smooth-bore barrel did not leave characteristic firing marks on the bullet.

Because of the enhanced intracranial tissue disruption (see companion paper) and the functional significance of the central nervous system, penetrating gunshot wounds of the head commonly result in immediate incapacitation. However, in the last century numerous publications reported sustained capability to act following penetrating gunshot wounds of the head. These are reviewed. A large number of case reports had to be excluded from re-examination because of doubtful capability to act or lack of morphological documentation. There remained 53 case reports from 42 sources for systematic analysis. Favourable conditions for sustained capability to act are present in cases where the additional wounding resulting from the special wound ballistic qualities of the head (see companion paper) are minimized. Thus, more than 70% of the guns used fired slow and lightweight bullets: 6.35 mm Browning, .22 rimfire or extremely ineffective projectiles (ancient, inappropriate or selfmade). A centre-fire rifle or a shotgun from close range were never employed in cases involving intracerebral tracts. A coincidence of several lucky circumstances made sustained capability to act possible in two cases of military centrefire rifle bullets passing longitudinally between the frontal lobes without direct contact with brain tissue. Only two large handguns resulting in intracerebral wounding were used: one firing a .38 special bullet, which solely wounded the base of the right temporal lobe and one firing a .45 lead bullet, which seriously injured the left frontal lobe but whose trajectory was limited to the anterior fossa of the skull. Of the trajectories, 28% were outside the neurocranium. At least 70% of the craniocerebral tracts passed above the anterior fossa of the skull, wounding the frontal parts of the brain. Apart from a neurophysiological approach, this preference can be explained by the fact that the base of the anterior cranial fossa and the sella turcica area serve as a bony barrier protecting the parts of the brain located in its "shadow"' relative to the trajectory against cavitational tissue displacement and associated overpressures. This is particularly true of the brain stem. Intracerebral trajectories not located above the anterior fossa were caused by slow and lightweight bullets preferring one temporal lobe. Additionally, one parietal and one occipital lobe were each injured once by a very ineffective projectile and by a 7.65-mm bullet reduced in velocity. Not a single case of injury to the brain stem, the diencephalon, the cerebellum or major paths of motor conduction and only one grazing shot of the anterior parts of the nucleus caudatus (basal ganglia) were described. Morphological signs of high intracranial pressure peaks (cortical contusion zones, indirect skull fractures, perivascular haemorrhages) and secondary missiles were poorly documented. It is suggested that these findings are at least very rare and not obvious in cases of sustained capability to act.

A highly selective electrophoretic system employing differential hydrophobic interaction was evaluated for the quantitative determination of recombinant insulin-like growth factor I (IGF-I) variants. The system consisted of mixed aqueous-organic buffers containing suitable amounts of a zwitterionic detergent. In addition, a neutral hydrophilic coating was attached to the wall of the capillary to minimize analyte adsorption and electroosmotic flow. The zwitterionic detergent acted as a hydrophobic selector, allowing independent optimization of the electrophoretic and hydrophobic selectivities in the separation system. The extent of hydrophobic interaction was conveniently adjusted by varying the type and amount of organic modifier. Complete resolution of a mixture of IGF-I variants with closely related mass-to-charge ratios was achieved. Quantitative analysis of IGF-I process samples agreed well with HPLC results. Finally, the approach was found to be compatible with on-line capillary electrophoresis-mass spectrometry.

Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.

A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' fragment of a mouse monoclonal antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled antibody fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence. In these preliminary results, methionyl recombinant human growth hormone (met-rhGH) could be successfully determined down to detection levels of approximately 5 x 10(-12) M. Mono- and dideamidated variants of met-rhGH were detected simultaneously with the nondeamidated form of the antigen as separate peaks.

Polyvinylmethylsiloxanediol (50% vinyl) was synthesized and combined with a cross-linker for static coating onto fused-silica columns. After cross-linking and binding to the surface, linear polyacrylamide was grafted to the double bonds of the siloxanediol; subsequently, this linear polymer matrix was cross-linked with formaldehyde. The grafted neutral polymeric layer provided suppression of electroosmotic flow and minimized adsorption. This combination yielded successful open tube and polymer network separations of proteins, peptides and DNA molecules. Very high efficiencies (ca. 1 x 10(6) plates/m) were achieved for open tube protein separations, and hundreds of consecutive runs were performed with minimal change in migration times.

Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32 degrees C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

The qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co-ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co-ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface-coated fused-silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 microL was injected into a 75 microns ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10(-7)-10(-8) M, with detection limits of approximately 10(-9) M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.