Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain.

Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.

A polar, solvent-exposed residue can be essential for native protein structure.

BACKGROUND: A large energy gap between the native state and the non-native folded states is required for folding into a unique three-dimensional structure. The features that define this energy gap are not well understood, but can be addressed using de novo protein design. Previously, alpha(2)D, a dimeric four-helix bundle, was designed and shown to adopt a native-like conformation. The high-resolution solution structure revealed that this protein adopted a bisecting U motif. Glu7, a solvent-exposed residue that adopts many conformations in solution, might be involved in defining the unique three-dimensional structure of alpha(2)D. RESULTS: A variety of hydrophobic and polar residues were substituted for Glu7 and the dynamic and thermodynamic properties of the resulting proteins were characterized by analytical ultracentrifugation, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. The majority of substitutions at this solvent-exposed position had little affect on the ability to fold into a dimeric four-helix bundle. The ability to adopt a unique conformation, however, was profoundly modulated by the residue at this position despite the similar free energies of folding of each variant. CONCLUSIONS: Although Glu7 is not involved directly in stabilizing the native state of alpha(2)D, it is involved indirectly in specifying the observed fold by modulating the energy gap between the native state and the non-native folded states. These results provide experimental support for hypothetical models arising from lattice simulations of protein folding, and underscore the importance of polar interfacial residues in defining the native conformations of proteins.

Acute and long-term cytogenetic effects of childhood cancer chemotherapy and radiotherapy.

Approximately 30 banded karyotypes per subject from the lymphocytes of 66 childhood cancer patients and 14 noncancer control subjects have been analyzed in an attempt to gauge the late effects of anticancer chemotherapy and chemotherapy plus radiotherapy on the genetic material, i.e., the chromosomes. The frequencies (f) of aberrant cells were: f = 1/306 among cells from noncancer controls; f = 1/377 from cancer patients prior to therapy, f = 1/15 from patients currently on chemotherapy; and f = 1/32 from posttherapy patients (range, 3 months to 22 years poattherapy). The frequency of chromosomally aberrant cells did not appear to change with time among posttherapy patients, and the majority of aberrations detected in subjects from this group were balanced rearrangements. This was not the case for the on-therapy group where unbalanced rearrangements and unstable aberrations predominated.

The interaction between RTX toxins and target cells.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type- and species-specific. These toxins offer interesting models for targeting, insertion and translocation of aqueous proteins into lipid membranes.

Amide exchange rates in Escherichia coli acyl carrier protein: correlation with protein structure and dynamics.

The acyl carrier protein (ACP) of Escherichia coli is a 77-amino acid, highly negatively charged three-helix protein that plays a central role in fatty acid biosynthesis. Previous NMR studies have suggested the presence of multiple conformations and marginally stable secondary structural elements. The stability of these elements is now examined by monitoring amide exchange in apo-ACP using NMR-based methods. Because ACP exhibits many rapid exchange rates, application of traditional isotope exchange methods is difficult. In one approach, heteronuclear correlation experiments with pulsed field-gradient coherence selection have reduced the time needed to collect two-dimensional 1H-15N correlation spectra to the point where measurement of exchange of amide protons for deuterium on the timescale of minutes can be made. In another approach, water proton selective inversion-exchange experiments were performed to estimate the exchange rates of protons exchanging on timescales of less than a second. Backbone amide protons in the region of helix II were found to exchange significantly more rapidly than those in helices I and III, consistent with earlier structural models suggesting a dynamic disruption of the second helix. Highly protected amides occur on faces of the helices that may pack into a hydrophobic core present in a partially disrupted state.

Serum leptin concentrations in Caucasian and African-American girls.

Because African-American girls are heavier, taller, and mature earlier than Caucasian girls, we hypothesized that the serum leptin concentration differs between the two groups. Serum leptin concentrations were measured by immunoassay in 12-h fasted blood samples collected from 79 Caucasian and 57 African-American girls between 8 and 17 yr of age. Body composition was measured by dual-energy x-ray absorptiometry, sexual maturity by physical examination, and physical fitness by treadmill testing. Serum leptin concentrations were positively correlated (P < 0.01) with maturation, body fatness, and insulin and were higher (6.6 ng/mL, P < 0.01) in the African-American girls after adjusting for age. The difference remained significant (P < 0.01) but was reduced to 3.2 ng/mL after controlling for differences in maturation, fat mass, and physical fitness. The higher serum leptin levels might play an important role in the accelerated growth and sexual maturation of African-American girls.

Pubertal African-American girls expend less energy at rest and during physical activity than Caucasian girls.

Between 1963 and 1991, the most dramatic increases in the prevalence of overweight in the United States have been reported in African-American girls. Lower basal energy expenditure and lack of physical activity are believed to be risk factors for excessive weight gain. We hypothesized that energy expenditure at rest and during physical activity are lower in pubertal African-American girls than in Caucasian girls. Basal metabolic rate and sleeping energy expenditure of 40 Caucasian and 41 African-American pubertal girls (matched for age, physical characteristics, body fat, and energy intake) were measured by whole-room calorimetry, energy expended for physical activity by the doubly labeled water method, sexual maturity by physical examination, body composition by dual-energy x-ray absorptiometry, physical fitness by treadmill testing, and energy intake by 3-day food record. After adjusting for soft lean tissue mass, the basal energy expenditure (1333 +/- 132 vs. 1412 +/- 132 kcal/day, P = 0.01) and energy expended for physical activity (809 +/- 637 vs. 1271 +/- 162 kcal/day, P < 0.01) were significantly lower in the African-American girls than in the Caucasian girls. The differences remained the same after controlling for differences in sexual maturity and/or physical fitness. The lower energy expenditure of the pubertal African-American girls suggests that they are at a higher risk of becoming overweight than their Caucasian counterparts.

Effect of the antiglucocorticoid RU486 on adrenal steroidogenic enzyme activity and steroidogenesis.

RU486, a synthetic steroid receptor antagonist, has strong antiprogesterone and antiglucocorticoid properties. Chronic RU486 administration in two patients with ectopic secretion of adrenocorticotropin (ACTH) has been associated with decreasing plasma cortisol concentrations. One explanation of this finding is that RU486 may directly inhibit adrenal steroidogenesis. To test this hypothesis, we measured the effect of RU486 on specific steroidogenic enzymatic steps using an in vivo rat and an in vitro monkey model. Hypophysectomized-castrated-ACTH-replaced Sprague-Dawley rats were given RU486 i.p. at daily doses of 0, 0.0005, 0.005, 0.05, 0.5 and 5 mg/kg body weight per day for 7 days. The animals were sacrificed, and blood and adrenal glands collected. Adrenal cortical mitochondria and microsomes were purified from the rats and from two untreated Cynomolgus macaque monkeys. Specific steroidogenic enzyme activities were measured in the rat by the incorporation of 14C-labeled steroid substrates into products. A similar protocol was used to assay the steroidogenesis in the monkey adrenal fractions in the presence and absence of added RU486. Although rat adrenal weights decreased significantly at the highest RU486 dose, plasma levels of corticosterone were similar in control and treated rats. Rat adrenal 3 beta-hydroxysteroid dehydrogenase/isomerase (3-HSD), 21-hydroxylase (21-OH) and 11-hydroxylase (11-OH) activities decreased with increasing RU486 doses, with 21-OH and 11-OH being most severely affected. Monkey adrenal 3-HSD, 21-OH, 11-OH, 17-hydroxylase and 17,20-desmolase similarly decreased in the presence of increasing in vitro concentrations of RU486.(ABSTRACT TRUNCATED AT 250 WORDS)

Recruitment and the manpower crunch.

In summary, the signals are clear that current trends indicate a scarcity of pathologists not too many years hence. Recruiting efforts must be mounted now to bring to the attention of the appropriate medical students that pathology is a demanding, rewarding clinical discipline whose importance and satisfaction reach beyond basic science and the physical confines of the clinical laboratory.

Medical uncertainty and the autopsy: occult benefits for students.

The autopsy has been of great importance in educating students regarding medical uncertainty. The marked decline in the use of the autopsy in medical education and continuing education has contributed significantly to the current discomfort among physicians regarding medical uncertainty and medical errors, which, in turn, has furthered the decline of the autopsy. Inordinate guilt, denial, and other defensive behaviors that many physicians marshall in response to uncertainty and error prevent these individuals from learning from their mistakes. The autopsy experience during medical school, properly utilized, helps students to confront fallibility and sets the stage for later successful management of uncertainty and error.

A population-based autopsy study of sudden, unexpected deaths from natural causes among persons 5 to 39 years old during a 12-year period.

All unexpected deaths in New Mexico from 1977 to 1988 were reviewed. By statute each such death must be reported to the Office of the Medical Examiner (OMI) and according to institutional policy autopsied even when death is presumed to be from natural causes. From this group the 650 index cases that form the basis of this report were obtained. The crude rate of sudden, unexpected death among New Mexico residents 5 to 39 years old during the study period was 6.6/100,000 persons at risk. As documented by autopsy, the underlying cause of death in a majority of these cases (53.4%) was related to cardiovascular disease and alcoholism. Male persons in general are at increased risk for sudden, unexpected death, and American Indian and black male persons are at greater risk than their Anglo and Hispanic counterparts. American Indians account for a disproportionate share of the unexpected deaths resulting from alcoholism, and black male persons are at particular risk for unexpected death resulting from cardiovascular diseases. This report emphasizes the importance of life style and diet in the well-being of persons 5 to 39 years old.

The current status of the autopsy in academic medical centers in the United States.

As viewed by pathology chairmen, the primary reasons for the decline in interest in the autopsy are: (1) a feeling among physicians that everything is known about the case; (2) poor education of medical students and clinical house staff concerning the importance of the autopsy, which carries over to the practitioner; and (3) lack of interest on the part of pathologists. Negative attitudes on the part of clinicians were seen as the primary factor that serves to inhibit enthusiasm for the autopsy on the part of pathology house staff. Lack of prompt and appropriate communication with the attending physician and uneven quality of prosectors are seen as major inhibitors to successful autopsy services. Chairmen of departments of pathology support an approximate doubling of the autopsy rate in their institutions (from 30% to 64%), although 42.5% of chairmen had not discussed their wishes concerning autopsy with the next of kin and only 42% regularly attend gross conference. These perceptions are remarkably similar to those provided by chairmen of departments of medicine and surgery as a part of a previous survey. On the basis of these inputs, several recommendations designed to improve the local emphasis on the autopsy service are provided.

Pathologists and the autopsy.

Autopsy practice today remains rooted in the fabric of medicine as it developed through the 18th and 19th centuries. Recent developments in medicine and society have left autopsy practice behind and have led to the decline of the autopsy. Potential new values of autopsies point strongly to the need for revitalized modern autopsy services, services focused on objectives and problems related to patients, their physicians, and the attendant societal issues. There are real values for pathology and pathologists, but only if major realignments in purposes and outcomes are forthcoming.

Reproducibility of three identification systems for biotyping of coagulase-negative staphylococci.

Three commercial identification systems were evaluated as tools for biotyping coagulase-negative staphylococci. Emphasis was placed on the reproducibility of component tests and not on the ability of these kits to identify these bacteria accurately. Forty-seven clinical and reference strains of Staphylococcus were tested in duplicate with each system. The Staph-Ident profile of test results changed for 20 strains on repeat testing, the Staph-Trac profile changed for 10 strains, and the Vitek GPI profile changed for 14 strains. The component tests of each system that were responsible for these profile changes were identified.

The effects of nonclassic pediatric bacterial pathogens on the usefulness of the Directigen latex agglutination test.

Haemophilus influenzae type b, Escherichia coli, Neisseria meningitis, Streptococcus agalactiae, and Streptococcus pneumoniae are classically the predominant meningeal pathogens of children. The Directigen latex agglutination test identifies these pathogens by detecting specific antigens in cerebrospinal fluid (CSF) and urine. The authors tested 1151 specimens from 791 children with suspected meningeal infections. They found that the sensitivity of the Directigen test for detecting the five classic CSF pathogens of children was 83.3% with CSF and 60% with urine specimens. In detecting all pathogens, however, the sensitivity was only 50% with CSF and 37.5% with urine. Thus, an increased prevalence of nonclassic pathogens in a pediatric population adversely affects the efficacy of the Directigen test for confirming a diagnosis of meningitis and emphasizes the diagnostic importance of the clinical history and other routine CSF tests.

Are basal metabolic rate prediction equations appropriate for female children and adolescents?

The basal metabolic rate (BMR), which accounts for 50-70% of total energy expenditure, is essential for estimation of patient and population energy needs. Numerous equations have been formulated for prediction of human BMR. Most equations in current use are based on measurements of Caucasians performed more than four decades ago. We evaluated 10 prediction equations commonly used for estimation of BMR in 76 Caucasian and 42 African-American girls between 8 and 17 yr of age against BMR measured by whole-body calorimetry. The majority of the prediction equations (9 of 10) overestimated BMR by 60 +/- 46 kcal/day (range, 15-176 kcal/day). This overestimation was found to be significantly greater (P < 0.05) for African-American (77 +/- 17 kcal/day) than for Caucasians (25 +/- 17 kcal/day) in six equations, controlling for age, weight, and sexual maturity. We conclude that ethnicity is an important factor in estimation of the BMR and that the current prediction equations are not appropriate for accurate estimation of the BMR of individual female children and adolescents.

Selection of low frequency tumor cells from cell culture by growth in nude mice.

Tissue cultures of tumor cells are frequently utilized to characterize chromosomal changes when direct cytogenetic preparations on tumors fail. The present study demonstrates that chromosomal markers found in direct tumor preparations can become undetectable in cell culture at variable rates presumably because of overgrowth of normal cell components in the culture. Injection of cultured tumor cells into nude mice followed by direct chromosomal preparations on the resulting nude mouse tumors can be used to select cells with the original tumor karyotype. This is true even when the tumor cell frequency in the culture is so low that they are not found in routine chromosomal preparations of the cultured cells. This technique can thus complement tissue culture findings and provide additional useful information about the original karyotype in cases where direct chromosomal preparations from tumors have failed.

Chromosome aberrations in vitro related to cytotoxicity of nonmutagenic chemicals and metabolic poisons.

Chromosome aberrations can occur by secondary mechanism(s) associated with cytotoxicity, induced by chemicals that do not attack DNA. Aberrations are formed from DNA double-strand breaks, and DSBs are known to be induced by nonmutagenic (Ames test negative) noncarcinogens at toxic levels [Storer et al. (1996): Mutat Res 368:59-101]. Here, 8 of 12 of these chemicals caused aberrations in CHO cells at cytotoxic doses, and often only when cell counts (survival) at 20 hr approached < or =50% of controls. Five of eight noncarcinogens (2,4,-dichlorophenol, dithiocarb, menthol, phthalic anhydride, and ethionamide) and one of two equivocal carcinogens (bisphenol A) caused aberrations, usually over a narrow dose range with steeply increasing cytotoxicity. Phthalic anhydride and ethionamide were positive only at doses with precipitate. Phenformin was negative even at toxic doses and ephedrine and phenylephrine were negative and gave little toxicity. Aberrations were also induced by metabolic poisons, 2,4-dinitrophenol, (uncouples oxidative phosphorylation), and sodium iodoacetate, (Nal; blocks ATP production). Five of the chemicals that induced aberrations in CHO cells were tested in human TK6 cells and four were positive, the fifth being equivocal. Stable aberrations (translocations) were induced in human cells by Nal. Clearly, chemicals can give "false-positive" results in the chromosome aberration assay at cytotoxic levels, though cytotoxicity does not always produce aberrations, so that further information (e.g., DNA reactivity) is needed to determine whether a result is a "false-positive." Primary DNA-damaging chemicals such as alkylators are also cytotoxic, but give strong increases in aberrations without marked initial toxicity by the measures used here, although the aberrations they induce do reduce long-term survival in colony-forming assays.

The uses and value of autopsy in medical education as seen by pathology educators.

A national meeting of pathology educators in 1989 provided the impetus for an exploration of new uses of autopsy in medical education. A month before the conference, the authors sent a questionnaire about the uses and value of autopsy in medical education to 120 persons registered to attend the conference. They used the 98 responses, representing 69 U.S. and Canadian medical schools, as the basis of a workshop on the place of autopsy in future medical education. The present article is a report of the authors' findings from the questionnaire and workshop. They found that the uses of autopsy go far beyond the traditional uses in teaching clinical pathophysiology, clinico-pathologic correlations, clinical anatomy, gross and microscopic anatomy of disease, and visual skills. Emphasis was placed on the potential role of autopsy in education regarding legal/judicial proceedings, vital statistics, epidemiologic investigations, and public health, and in the understanding of such complex matters as medical fallibility, medical uncertainty, and grief. These purposes were seen as congruent with current societal concerns about the need to reverse the trend toward dehumanization of medicine and physicians. The inability to realize these aims in the face of a precipitous drop in the autopsy rate is discussed.