The relevance of the evolution of experimental studies for the interpretation and evaluation of some trace physical evidence.

In order for trace evidence to have a high evidential value, experimental studies which mimic the forensic reality are of fundamental importance. Such primary level experimentation is crucial to establish a coherent body of theory concerning the generation, transfer and persistence of different forms of trace physical evidence. We contend that the forensic context, at whatever scale, will be specific to each individual forensic case and this context in which a crime takes place will influence the properties of trace evidence. It will, therefore, be necessary in many forensic cases to undertake secondary level experimental studies that incorporate specific variables pertinent to a particular case and supplement the established theory presented in the published literature. Such studies enable a better understanding of the specific forensic context and thus allow more accurate collection, analysis and interpretation of the trace physical evidence to be achieved. This paper presents two cases where the findings of secondary level experimental studies undertaken to address specific issues particular to two forensic investigations proved to be important. Specific pre-, syn- and post-forensic event factors were incorporated into the experimental design and proved to be invaluable in the recovery, analysis and in achieving accurate interpretations of both soil evidence from footwear and glass trace evidence from a broken window. These studies demonstrate that a fuller understanding of the specific context within which trace physical evidence is generated and subsequently collected, as well as an understanding of the behaviour of certain forms of trace physical evidence under specific conditions, can add evidentiary weight to the analysis and interpretation of that evidence and thus help a court with greater certainty where resources (time and cost) permit.

The impact of HBV-DNA fluctuations on virus-specific CD8+ T cells in HBeAg+ chronic hepatitis B patients treated with a steroid and lamivudine.

Restoration of anti-viral immune response may be a requisite for sustained virological response to treatment in chronic hepatitis B patients. Over a 13-month period, we examined the dynamics of hepatitis B virus (HBV)-specific CD8+ cells in six human leucocyte antigen (HLA)-A2+ hepatitis B e antigen (HBeAg)+ 'immunotolerant' chronic hepatitis B patients treated sequentially with corticosteroid and lamivudine. Our results show that the combination treatment did not result in a sustained restoration of anti-viral specific CD8+ cells in five of the six patients studied. However, HBV-specific CD8+ cells, despite being severely compromised, were not totally deleted. Paradoxically, steroid treatment was not associated with inhibition but with a minimal increase of the HBV-specific CD8 response, and we observed that nucleocapsid-specific CD8 responses were not rescued by stable and prolonged inhibition but became detectable after rapid rebounds of HBV replication. In most patients, the transient and minimal restoration of HBV-specific immunity was not associated with clinical benefits. Our results describe a dynamic relationship between HBV-specific CD8+ cells and HBV-DNA values, that could potentially be used for a better design of HBV treatment in HBeAg+ 'immunotolerant' chronic hepatitis B patients.

Sensorimotor adaptations to microgravity in humans.

Motor function is altered by microgravity, but little detail is available as to what these changes are and how changes in the individual components of the sensorimotor system affect the control of movement. Further, there is little information on whether the changes in motor performance reflect immediate or chronic adaptations to changing gravitational environments. To determine the effects of microgravity on the neural control properties of selected motor pools, four male astronauts from the NASA STS-78 mission performed motor tasks requiring the maintenance of either ankle dorsiflexor or plantarflexor torque. Torques of 10 or 50% of a maximal voluntary contraction (MVC) were requested of the subjects during 10 degrees peak-to-peak sinusoidal movements at 0.5 Hz. When 10% MVC of the plantarflexors was requested, the actual torques generated in-flight were similar to pre-flight values. Post-flight torques were higher than pre- and in-flight torques. The actual torques when 50% MVC was requested were higher in- and post-flight than pre-flight. Soleus (Sol) electromyographic (EMG) amplitudes during plantarflexion were higher in-flight than pre- or post-flight for both the 10 and 50% MVC tasks. No differences in medial gastrocnemius (MG) EMG amplitudes were observed for either the 10 or 50% MVC tasks. The EMG amplitudes of the tibialis anterior (TA), an antagonist to plantarflexion, were higher in- and post-flight than pre-flight for the 50% MVC task. During the dorsiflexion tasks, the torques generated in both the 10 and 50% MVC tasks did not differ pre-, in- and post-flight. TA EMG amplitudes were significantly higher in- than pre-flight for both the 10 or 50% MVC tasks, and remained elevated post-flight for the 50% MVC test. Both the Sol and MG EMG amplitudes were significantly higher in-flight than either pre- or post-flight for both the 10 and 50% MVC tests. These data suggest that the most consistent response to space flight was an elevation in the level of contractions of agonists and antagonists when attempting to maintain constant torques at a given level of MVC. Also, the chronic levels of EMG activity in selected ankle flexor and extensor muscles during space flight and during routine activities on Earth were recorded. Compared with pre- and post-flight values, there was a marked increase in the total EMG activity of the TA and the Sol and no change in the MG EMG activity in-flight. These data indicate that space flight, as occurs on shuttle missions, is a model of elevated activation of both flexor and extensor muscles, probably reflecting the effects of programmed work schedules in flight rather than a direct effect of microgravity.

Self-report evaluation of health behavior, stress vulnerability, and medical outcome of heart transplant recipients.

OBJECTIVE: The purpose of this study was to explore the value of patient self-report assessment in heart transplant candidacy evaluation, utilizing the Millon Behavioral Health Inventory (MBHI). Patient's MBHI measures were related to important pretransplant patient characteristics and posttransplant measures of health behavior, medical morbidity, and mortality. METHOD: Ninety heart patients with end-stage cardiac disease completed the MBHI during pretransplant candidacy evaluations, and also were interviewed concerning their coping effectiveness, support resources, and compliance history. Postransplant follow-up of 61 living and 29 deceased patients included measures of survival time, postsurgical medical care, rejection and infection episodes, and nurse ratings of medication compliance and problematic interpersonal health behaviors. RESULTS: The MBHI coping scales were found to significantly discriminate good and poor pretransplant compliance, and interview judgments of good and poor coping and support resources, with modest accuracy. The MBHI also was superior to these interview judgments in predicting posttransplant survival time and medical care used. Certain scales were also positively associated with physical parameters of pretransplant and posttransplant status. CONCLUSIONS: Patient self-report with the MBHI can contribute to identification of patients at risk for a problematic outcome with transplant, by providing information pertinent to clinical decision making and outcome management analysis with this special population of cardiac patients.

Psychiatric interview and psychometric predictors of cardiac transplant survival.

OBJECTIVE: This study prospectively investigated the relation between pretransplant assessment of psychiatric diagnosis, coping skills, and social support and outcome measures of survival and health care utilization. METHOD: Ninety-four heart transplant patients underwent structured interviews and completed a battery of self-report psychometric tests assessing coping style, health status, and psychosocial supports. Follow-up that ranged from 9 to 56 months after transplant produced a group of 63 survivors and 31 nonsurvivors, who were found to be comparable in terms of pretransplant cardiac status and severity of illness. RESULTS: Survival analysis showed that dimensional psychometric measures of coping and social support based on patient self-assessment were the best significant predictors of survival. Considered separately, interview-determined ratings of social support and pretransplant compliance with treatment regimens were also potential predictors. Formal DSM-III-R diagnoses were related specifically to posttransplant hospital utilization (axis I diagnoses) and posttransplant health behavior (axis II diagnoses). CONCLUSIONS: The data demonstrate the value of multifaceted assessment, since psychiatric diagnosis, coping style, and psychosocial support may contribute differently to prediction of such aspects of outcome as mortality and health care utilization.

Biochemical and immunological properties of two forms of pertactin, the 69,000-molecular-weight outer membrane protein of Bordetella pertussis.

Two apparent isoforms of the virulence-associated 69,000-molecular-weight protein pertactin were purified from Bordetella pertussis. Mass spectrometry showed a difference of 2,060 Da, which may result from differential C-terminal cleavage of a larger precursor. Both forms were protective in a mouse model, eliciting bactericidal antibodies and reducing respiratory tract colonization.

Evaluation of antibodies elicited by immunization with pertussis toxin.

The monoclonal antibody termed 1B7 neutralizes pertussis toxin in vivo in cell culture systems and can also passively protect mice from a challenge with live Bordetella pertussis (9). It has been suggested that most other independently derived neutralizing monoclonal antibodies recognizing the S1 subunit apparently recognize the same epitope as 1B7, and that the S1 subunit contains only one immunodominant protective epitope (1). These antibodies have been termed Class A antibodies (8) and inhibit the ADP-ribosyltransferase but not the NAD glycohydrolase activity of the toxin (7). We are testing the hypothesis that immunization with inactivated preparations of pertussis toxin that lead to protection are associated with the production of Class A antibodies. If true, then identification of Class A antibodies in sera might provide a serological correlate of protection. If false, then development of assays designed to detect the important protective antibodies are necessary. Our initial results suggest that Class A antibodies are not the predominant neutralizing antibody in mice immunized with vaccines containing formalin-treated pertussis toxin.

Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis.

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.

Regulation of nitrogen fixation in Rhodospirillum rubrum grown under dark, fermentative conditions.

Rhodospirillum rubrum was shown to grow fermentatively on fructose with N2 as a nitrogen source. The nitrogenase activity of these cells was regulated by the NH4+ switch-off/switch-on mechanism in a manner identical to that for photosynthetically grown cells. In vitro, the inactive nitrogenase Fe protein from fermenting cells was reactivated by an endogenous membrane-bound, Mn2+-dependent activating enzyme that was interchangeable with the activating enzyme isolated from photosynthetic membranes.

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum.

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH4+. Activity of the Fe protein component of nitrogenase required both Mn2+ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.

Purification and Mn2+ activation of Rhodospirillum rubrum nitrogenase activating enzyme.

The Fe protein activating enzyme for Rhodospirillum rubrum nitrogenase was purified to approximately 90% homogeneity, using DE52-cellulose chromatography and sucrose density gradient centrifugation. Activating enzyme consists of a single polypeptide of molecular weight approximately 24,000. ATP was required for catalytic activity, but was relatively ineffective in the absence of Mg2+. When the concentration of MgATP2- was held in excess, there was an additional requirement for a free divalent metal ion (Mn2+) for enzyme activity. Kinetic experiments showed that the presence of Mg2+ influenced the apparent binding of Mn2+ by the enzyme, resulting in a lowering of the concentration of Mn2+ required to give half-maximum activity (K alpha) as the free Mg2+ concentration was increased. A low concentration of Mn2+ had a sparing effect on the requirement for free Mg2+. There is apparently a single metal-binding site on activating enzyme which preferentially binds Mn2+ as a positive effector, and free Mg2+ can compete for this site.

Effect of light intensity and inhibitors of nitrogen assimilation on NH4+ inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp.

Nitrogenase activity in Rhodospirillum rubrum was inhibited by NH4+ more rapidly in low light than in high light. Furthermore, the nitrogenase of cells exposed to phosphorylation uncouplers was inhibited by NH4+ more rapidly than was the nitrogenase of controls without an uncoupler. These observations suggest that high levels of photosynthate inhibit the nitrogenase inactivation system. L-Methionine-DL-sulfoximine, a glutamine synthetase inhibitor, prevented NH4+ from inhibiting nitrogenase activity, which suggests that NH4+ must be processed at least to glutamine for inhibition to occur. An inhibitor of glutamate synthase activity, 6-diazo-5-oxo-L-norleucine, inhibited nitrogenase activity in the absence of NH4+, but only in cells exposed to low light. The mechanism of 6-diazo-5-oxo-L-norleucine inhibition appeared to be the same as that induced by NH4+, because nitrogenase activity could be restored in vitro by activating enzyme and Mn2+. The inhibitor data suggest that the glutamine pool or a molecule that responds to it activates the Fe protein-modifying (or protein-inactivating) system and that the accumulation of this (unidentified) molecule is retarded when the cells are exposed to high light. It was confirmed here that Anabaena nitrogenase is also inhibited by NH4+, but only when the cells are incubated under low light. This inhibition, however, unlike that in R. rubrum, could be completely reversed in high light, suggesting that the mechanisms of nitrogenase inhibition by NH4+ in these two phototrophs are different.

Novel mutant of Anabaena sp. strain CA which growns on N2 but not on combined nitrogen.

A mutant has been isolated from Anabaena sp. strain CA by treatment with N-methyl-N'-nitro-N-nitrosoguanidine, which has the unusual phenotypic characteristic of growth only under N2-fixing conditions. Growth of the mutant was completely inhibited by NO3- or NH4+ at concentrations routinely used for growth of the wild type, and sensitivity to NH4+ was especially pronounced. The inhibitory effect of NH4+ could not be overcome by glutamine, glutamate, or casein hydrolysate. Ammonia had no immediate inhibitory effect on protein synthesis, CO2 fixation, or O2 evolution, and the gradual inhibition of C2H2 reduction activity by NH4+ resembled a repression phenomenon. The glutamine synthetase activity of N2-fixing cultures appeared normal, yet the mutant was incapable of utilizing exogenous NH4+ for growth. Preliminary evidence suggests a possible alteration of glutamine synthetase, which could result in sensitivity to exogenous NH4+ by progressive inactivation of the enzyme or repression of its synthesis.

Mutants of Anabaena strain CA altered in their ability to grow under nitrogen-fixing conditions.

Mutants of Anabaena strain CA impaired in nitrogenase activity and growth on N2 were isolated and characterized. One mutant was selected for resistance to L-methionine-DL-sulfoximine, and others were selected for resistance to DL-7-azatryptophan or for requirements for combined nitrogen. The mutants varied in sensitivity of growth and nitrogenase activity to atmospheric 02. Several of the mutants whose growth on N2 was impaired under aerobic conditions could grow and reduce acetylene at rates comparable to the wild type when grown microaerobically under N2-CO2 (99:1). The acetylene reduction activity of some of the strains grown under N2-CO2 was immediately and completely lost upon exposure to atmospheric O2, but in at least one strain this loss was reversed when the O2 concentration was lowered, even after 10 h of exposure to air. The characteristics of the O2-sensitive mutants suggest that there may be several sites sensitive to O2 and that the protective mechanism involves several different phenomena.

N(2) Fixation Associated with Decaying Leaves of the Red Mangrove (Rhizophora mangle).

N(2) (C(2)H(2)) fixation was associated with decaying leaves of Rhizophora mangle. The process was predominantly anaerobic, with about two-thirds of the nitrogenase activity being light dependent. Average N(2) fixation rates in the light were 11 mug of N per g (dry weight) per h for leaves that had decayed for 2 to 3 weeks. This nitrogen input is probably significant in the estuarine, detrital food chains linked to R. mangle.