The evolution of a faculty development program in an academic psychiatry department.

Issues affecting faculty development in academic psychiatry departments have taken on a special urgency as a result of declining financial support from government sources and the emerging consequences of this country's fast-evolving health care system. Junior faculty need usable information on how to succeed in academia. Available information from the senior administration ("top-dawn") often lacks the immediacy and accessibility of grassroots ("bottom-up") strategies that more experienced junior colleagues have discovered. Over the past 2 years, the Department of Psychiatry at the University of Texas-Houston Health Science Center has focused attention on codifying these grassroots efforts and on integrating them with formal departmental initiatives. The authors describe how general recommendations formulated by departmental planning committees and a Continuous Quality Improvement Team on faculty development were developed into a formal, concrete program f or career development. (Academic Psychiatry 1997; 21:107-115).

Interspecific variation at the Y-linked RPS4Y locus in hominoids: implications for phylogeny.

Within- and between-species variation in restriction endonuclease recognition sites was examined at the Y-linked RPS4Y locus of six hominoid species: human (Homo sapiens), gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), orangutan (Pongo pygmaeus), and gibbon (Hylobates lar). RPS4Y is an expressed gene that maps to the non-recombining region of the Y chromosome. An approximately 1,490 base pair fragment of the RPS4Y gene, including all of intron 3, was amplified by PCR from DNA extracted from each of the six species. Forty-seven restriction sites were identified on the six-species composite map derived from double-digest restriction analyses of the amplified fragment. As expected, maximum parsimony analysis indicated that chimpanzee and bonobo are the two most closely related living hominoids. The same analysis suggested that the closest living relative of Homo is Gorilla, not Pan, although support for this relationship was relatively weak. These results disagree with recently published phylogenies based on analyses of mtDNA sequences (Horai et al. [1995] Proc. Natl. Acad. Sci. U.S.A. 88:7401-7404) and the Y-linked ZFY locus (Dorit et al. [1995] Science 268:1183-1185). A combined data set derived from three distinct Y-linked loci-RPS4Y, SRY, and ZFY-was also analyzed. The maximum parsimony topology for the combined data provided only weak support for a shared common ancestor for Homo and Pan subsequent to divergence from the Gorilla lineage. Taken together, the data from the Y chromosome do not provide unequivocal support for any single, dichotomously branching species tree linking Homo, Pan, and Gorilla.

A new DNA marker, U15557, is linked to protease inhibitor and adenylate kinase-1 in the laboratory opossum, Monodelphis domestica.

A 323-bp DNA fragment (U15557) was isolated, cloned, and sequenced after polymerase chain reaction (PCR) amplification from Monodelphis domestica genomic DNA. A HindIII restriction fragment length polymorphism was identified in this species using the U15557 PCR, fragment as a hybridization probe. DNA samples exhibited either a 6.4 kb band, a 7.2 kb band, or both bands simultaneously. Behaviour of these two variants in family studies was consistent with codominant autosomal inheritance. Linkage between this marker and the loci encoding protease inhibitor (PI) and adenylate kinase 1 (AK1) was found in M. domestica.

Age, gender, and sensitivity to bleomycin-induced chromosome breakage in Down's syndrome and control populations.

The free radical theory of aging suggests that cells from individuals with premature aging syndromes, such as Down's syndrome, might be especially sensitive to radical-induced DNA damage. Although existing studies support this hypothesis, little work has been done on examining the impact of increasing age on radical sensitivity within the Down's syndrome population. Additionally, intercellular heterogeneity and gender differences in radical sensitivity have not been investigated. In this study, bleomycin-induced chromosome breakage was measured in lymphocytes from a population of adult men and women with Down's syndrome (age range 20-60 years) and a population of normal control men and women of the same age range. Change in breakage rates as a function of age and intercellular heterogeneity in breakage rates were examined in males and females of both groups. The findings are discussed in connection with longevity and cancer risk in the old male population.

Characterization of the DiFi rectal carcinoma cell line derived from a familial adenomatous polyposis patient.

The DiFi human colorectal cancer cell line was recently established from a familial adenomatous polyposis patient with extracolonic features characteristic of the Gardner syndrome. These cells have now been propagated for 150 passages in standard culture media and vessels without feeder layers or collagen coatings. They retain features of colonic epithelial cells such as surface microvilli, secretory vesicles, and desmosomes. Cytosol of DiFi cells contains a high level (502 U/mg protein) of the mucin CA 19-9. In addition, DiFi cells produce carcinoembryonic antigen, and induce tumors in athymic mice. Cytoskeleton analysis of DiFi cells by fluorescence microscopy showed a pronounced disorganization of actin cable structure. The isozyme genetic signature of DiFi cells is unique (0.01 probability of finding the same genetic signature in a different cell line), differs from that of HeLa cells, and has expressional features seen in other colorectal cell lines. The DiFi cell karyotype is tetraploid, contains many marker chromosomes, and shows numerous episomal particles. Two copies of chromosome 18 were absent, and only a single normal chromosome 17 was found. This parallels detection of allelic losses from DiFi cell DNA at loci on chromosomes 17p and 18 using molecular (cDNA) probes. DiFi cells clearly express transcripts for the c-myc proto-oncogene, the c-myb proto-oncogene, and the p53 tumor suppressor gene. Transforming growth factor beta inhibits DiFi cell growth in soft agar and suppresses c-myc expression in these cells. The value of this cell line in the study of genetic alterations in colorectal cancer is discussed.

Gender differences and the interpretation of genetic instability in Alzheimer's disease.

The neuronal degeneration and death which characterize Alzheimer's disease (AD) may stem from a constitutive genetic instability related to DNA repair deficits. To test this hypothesis, we treated peripheral blood lymphocytes from persons with AD, age-matched controls, and young controls with two drugs that induce chromosome breakage. Bleomycin, a radiomimetic antineoplastic drug, causes single- and double-stranded DNA breaks through the generation of activated oxygen radicals. Methyl methane-sulfonate (MMS) is a monofunctional alkylating agent that binds covalently to DNA. Cells were grown in culture for 72 h, with drug treatments for 4 h (bleomycin) or 24 h (MMS) prior to harvest. Fifty cells per subject per drug were scored for chromosome breakage. Breakage rates for both drugs in AD women were significantly higher than those in age-matched control women. This was not the case in men, due to the very high induced breakage rates seen in the age-matched normal control men. Because the induced breakage rates in AD women and AD men are equivalent, it seems likely that an independent factor may be contributing to genetic instability in the normal control men. Our findings indicate that the interpretation of the response of AD lymphocyte chromosomes to DNA-damaging chemicals can be strongly confounded by the effects of gender ratio in the control population sampled. These findings have important implications for the design of future studies of Alzheimer's disease, as well as for the assessment of health risks in unaffected elderly populations.

Mitosis-arresting effects of cigarette smoke condensate on human lymphoid cell lines.

Whole-smoke condensates from the University of Kentucky reference cigarettes induced partial mitotic arrest in 4 human lymphoid cell lines. Treatment of cells for 3 h with 100 and 200 micrograms of cigarette-smoke condensate/ml of culture medium increased the frequency of metaphases and decreased the proportion of anaphases in the treated cell populations. Cytoskeletal studies using antitubulin immunofluorescence techniques and transmission electron microscopic studies demonstrated that in early stages of mitosis the formation of aster and the separation of centrosomes in condensate-treated cells were comparable to those of untreated control cells, but the poleward migration of centrosomes was inhibited. Arrested metaphases revealed two centrosomes surrounded by aggregated chromosomes in the center of each cell but the structure of the centrioles, microtubules and the kinetochores appeared normal. The results demonstrate the presence of antimitotic compounds in the tobacco-smoke condensate.

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity.

Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations.

Sensitivity to genotoxic effects of bleomycin in humans: possible relationship to environmental carcinogenesis.

Responses to the genotoxic effect of bleomycin in lymphocytes of blood cultures, expressed as the average number of chromatid breaks per cell (b/c), varied from less than 0.20 to more than 2.00 in 335 normal individuals. More than 11% of the subjects tested showed a b/c rate above 1.00 and more than 22% showed a b/c rate above 0.80. These individuals are considered sensitive to this radiomimetic drug. The distributional profile of bleomycin responses of the control individuals appears to be representative of the normal human population. In patients with cancers of the colon (83), upper aerodigestive tract (head/neck) (77), and lung (71), the frequencies of subjects in the hypersensitive class were found to be between 40 and 50%, and the response profiles were distinctly different from those of the control population. On the other hand, in a group of elderly cigarette smokers, who exhibited no symptoms of lung cancer, the bleomycin sensitivity profile was significantly skewed toward the more resistant stratum, with only one hypersensitive case among 56 individuals tested (1.78%). The sensitivity profile of patients with breast cancer (82) was similar to that of the control population. Our data suggest that: (1) mutagen sensitivity may play an important role in carcinogenesis of organs and tissues that have direct contact with the external environment (respiratory, digestive, and integumentary systems); (2) it appears to have no significant influence on carcinogenesis of tissues that are not directly exposed to the environment (e.g., breast, brain); and (3) it also has little impact on carcinogenesis in individuals with a hereditary predisposition to cancer (e.g., retinoblastoma, Gardner's syndrome). Development of more effective and precise test systems for carcinogen sensitivity is highly desirable for identification of persons at risk.

Kinetochore size variation in mammalian chromosomes: an image analysis study with evolutionary implications.

The kinetochore, a proteinaceous plate that is the site for attachment of spindle microtubules to the metaphase chromosome, can be visualized using anti-kinetochore indirect immunofluorescence. We have used computer-assisted image analysis to measure the variation of kinetochore surface areas, as reflected by immunofluorescence areas, in cell lines derived from rat kangaroo, Chinese hamster and common rat, to determine if our size estimates correlate well with those obtained using measurements from electron micrographs. In addition, we used male and female human fibroblast cell lines, as well as a transformed human female cell line as well as a transformed human female cell line (HeLa), to examine kinetochore size variation among cells, between sexes, and between cell lines. We found that our system gave reproducible estimates of kinetochore size, and that these sizes correlated very well (r = 0.95) with the electron micrograph measurements. In examining variation within humans, we observed measurable differences between cell lines. Despite this difference, all the human lines had size distributions that were leptokurtotic and positively skewed. The fact that very few chromosomes exhibited areas smaller than the mode gives support to the idea that mammalian chromosomes may require a specific, minimum amount of kinetochore material in order to maintain stable attachment to the mitotic spindle. On the other hand, the positive skewness seems to indicate that larger kinetochores, possibly the result of events such as Robertsonian fusions, are fully functional. The retention of this plasticity may allow the chromosomes to maintain an evolutionary adaptability that might otherwise be lost.

Nuclear and manchette development in spermatids of normal and azh/azh mutant mice.

Germinal cells or nuclei with attached cytoskeletal elements were prepared from the testes and epididymides of normal mice and mice homozygous for the recessive azh mutation, which results in abnormal sperm heads. To make observations, we utilized phase-contrast microscopy, immunofluorescence microscopy with antitubulin antibodies, and a direct-view stereo electron microscope system developed by A. Cole. Sperm nuclei, tails, manchettes, and other cytoskeletal structures were studied at various stages of development. The tail architectures were similar in the normal and mutant forms, but the shape of the heads at the attachment regions were markedly different. Normal sperm nuclei were very flat, whereas the posterior regions of mutant nuclei were tapered cylinders. The manchette, an organized microtubular structure that girdles the posterior region of the spermatid nucleus, differed in size and configuration between normal and mutant forms. In normal midstage spermatids, the manchette microtubules extended outward at a 45 degree angle from the long axis of the flattened head, whereas in mutant spermatids, the microtubules formed tapered cylinders around the long axis of the caudal part of the nucleus. Radical differences in head shapes between normal and mutant sperm could be related, in part, to the manner in which manchettes formed and matured on the spermatids.

Kinetochore staining in multicentric chromosomes of murine cancer cell lines.

The production of multicentric chromosomes is a common event in neoplastic or evolutionary change. If these compound chromosomes are to remain intact, the microtubule-binding ability of one of the kinetochores must be inactivated. This inactivation could occur by an actual deletion of chromosome material, or by some conformational change which would serve to interrupt microtubule binding. To answer this question, staining techniques are required which are specific for structural elements contained in the kinetochore. Stable compound chromosomes were studied in two murine cancer cell lines to see if there is concordance among the currently available light microscope techniques reported to stain the kinetochore. It was discovered that many commonly used approaches give no direct information about the presence of kinetochore activity or kinetochore structural elements. This information is available, however, using antikinetochore antibody immunofluorescence. The method demonstrates that kinetochore inactivation is a complex and gradual event, involving the loss of at least some of the kinetochore-specific proteins.

A technique for simultaneous antikinetochore immunofluorescence staining and Q-banding in chromosomes from human lymphocytes.

Antikinetochore immunofluorescence staining has been used in several studies to determine whether a second kinetochore is present, active, or both, in multicentric chromosomes. All of these studies have used tissue culture cells, and contended with the problem of obtaining well spread, banded metaphase chromosomes without affecting the kinetochore staining. We have adapted hypotonic, centrifugation and chromosome staining techniques to obtain simultaneous Q-banding and bright kinetochore staining of chromosomes from human lymphocytes.

Size variation in kinetochores of human chromosomes.

Aneuploidy, the loss or gain of chromosomes from cells, is likely in many cases to involve the kinetochore, the site of attachment of spindle microtubules. We analyzed human fibroblast cells with antikinetochore-antibody indirect immunofluorescence, and noted an apparent heterogeneity in the sizes of kinetochores among different chromosomes. The Y chromosome in particular always showed minute kinetochores, an observation which was quantified and substantiated using computer-assisted image analysis. This finding, combined with literature reports about in vivo and in vitro involvement of the Y chromosome in aneuploidy, was used to frame a novel hypothesis about the generation of chromosome imbalance.

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro.

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.

Anti-tubulin immunofluorescence studies of recovery from vinblastine cytotoxicity in Chinese hamster cells.

Antitubulin immunofluorescent staining was used to examine the relationship among crystal formation, mitotic arrest, and recovery potential in vinblastine-treated Chinese hamster cells. Although vinblastine caused a mitotic block at concentrations as low as 5 x 10(-9) M, it induced tubulin crystal formation only at concentrations higher than 10(-6) M. At these higher concentrations, cells took 48-72 h to recover after return to normal medium. This extended period of time was apparently needed for breakdown of the crystals and regeneration of normal cytoplasmic microtubules. At concentrations less than 10(-6) M, although the mitotic block was still effective, no crystals were present. Possibly because of this lack of crystal formation, the cells recovered rapidly, generating cytoplasmic microtubules within 30 min, and beginning to undergo mitosis within 60 min. These findings tend to support biochemical evidence that tubulin binds to vinblastine at two types of binding site: a high affinity, low capacity site, responsible for tubulin disaggregation; and a low affinity site, responsible for protofilament splaying.

Differential mutagen susceptibility in cultured lymphocytes of normal individuals and cancer patients.

When cultured human lymphocytes were treated with a radiomimetic chemical bleomycin, metaphase chromosomes from different individuals exhibited a wide spectrum of responses in terms of number of chromatid lesions. This variability is interpreted as the result of capability for DNA repair. Among 100 healthy, normal individuals tested, nearly 60% showed a mean breaks per cell rate in the range of 0.20-0.60, whereas, only 12% showed breaks per cell rates above 1.00. On the other hand, among 75 cancer patients, 60% showed breaks per cell rates above 1.00. It is believed that differential responses to a mutagen have genetic implications. Individuals with DNA repair deficiencies may be more susceptible to carcinogens and, therefore, are more susceptible to neoplastic induction.