Much ado about cloning in the public square.

The dawnings of the age of human cloning and genetics is shaping lives, ideologies, and social structures. How will we--as individuals and as communities--respond to the possibilities, challenges, and changes of the clone age? This essay invites engagement in communal moral deliberation through broadening conversations about serious matters, including human cloning. A framework that includes important moral markers for significant "kitchen table talk" is offered. Clone age justice is also discussed. Attention is paid to the renewed role of religious voices in the public square.

Redox cycling of 2-(x'-mono, -di, -trichlorophenyl)- 1, 4-benzoquinones, oxidation products of polychlorinated biphenyls.

Polychlorinated biphenyl (PCB) preparations are complete liver carcinogens in rodents and efficacious promoters in two-stage hepatocarcinogenesis. Cytochrome P450 isozymes catalyze the oxidation of PCBs to mono- and dihydroxy metabolites. The potential for further enzymatic or nonenzymatic oxidation of ortho- and para-dihydroxy PCB metabolites to (semi)quinones raises the possibility that redox cycling involving reactive oxygen species may be involved in PCB toxicity. Seven synthetic 2-(x'-chlorophenyl)-1, 4-benzoquinones (containing one to three chlorines) were investigated for their participation in oxidation-reduction reactions by following the oxidation of NADPH. These observations were made: (i) NADPH alone directly reduced all quinones but only 2-(2'-chlorophenyl)- and 2-(4'-chlorophenyl)-1,4-benzoquinone supported NADPH consumption beyond that required to quantitatively reduce the quinone. (ii) For all quinones, superoxide dismutase increased NADPH oxidation in excess of the amount of quinone, demonstrating the participation of the superoxide radical. (iii) The presence of microsomal enzymes from rat liver increased the rate of NADPH consumption, but only 2-(2'-chlorophenyl)- and 2-(4'-chlorophenyl)-1,4-benzoquinone autoxidized. (iv) The combination of superoxide dismutase with microsomal enzymes accelerated autoxidation from 1.6- to 6.8-fold higher than that found in the absence of microsomal protein. These data support the concept that in the absence of microsomal protein, there occurs a two-electron reduction of the quinone by NADPH to the corresponding hydroquinone that comproportionates with the large reservoir of quinone to initiate autoxidation. In the presence of microsomes, enzymatic one-electron reduction generates a semiquinone radical whose autoxidation with oxygen propagates the redox cycle. These results show the potential of some 2-(x'-chlorophenyl)-1, 4-benzoquinones to initiate the wasteful loss of NADPH.

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

Yeast pre-mRNA splicing requires a pair of U1 snRNP-associated tetratricopeptide repeat proteins.

The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA. Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP. In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties. Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p. Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events. Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42 fusion gene arrest for splicing prior to pre-mRNA 5' splice site cleavage. Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S. cerevisiae (28). Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts. Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro. The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl.

Polychlorinated biphenyls (PCBs) may be metabolically activated to electrophiles, which bind to proteins and nucleic acids. One activation scheme involves the formation of reactive arene oxide intermediates during cytochrome P450-catalyzed hydroxylation. We propose a second activation pathway whereby PCB catechol and hydroquinone metabolites may be oxidized to reactive semiquinones and/or quinones. By employing 4-monochlorobiphenyl (4-MCB) as a model substrate and liver microsomes from rats treated with phenobarbital and 3-methyl-cholanthrene, five monol and three diol metabolites were identified. The major metabolite was 4-chloro-4'-monohydroxybiphenyl, followed by, in decreasing order, 4-chloro-3',4'-dihydroxybiphenyl, unknown B (a monol), 4-chloro-2',3'-dihydroxybiphenyl, 4-chloro-3'-hydroxybiphenyl, 4-chloro-2',5'-dihydroxybiphenyl, unknown A (a monol), and 4-chloro-2'-monohydroxybiphenyl. A trace of a dihydrodiol was detected by GC/MS. To elucidate the source of the diols, 4-MCB and the synthetic monol metabolites 4-chloro-2'-/-3'-/-4'-monohydroxybiphenyls were each employed as substrates in incubations with microsomes from rats treated with phenobarbital, 3-methylcholanthrene, or both inducers. The three diol metabolites were all produced from 4-MCB in incubations with microsomes from 3-methylcholanthrene-treated rats, but incubations with microsomes from phenobarbital-treated rats did not yield detectable amounts of 4-chloro-2',3'-dihydroxybiphenyl. 4-Chloro-2',3'-dihydroxybiphenyl was only found as a product of 4-chloro-2'-monohydroxybiphenyl. The 4-chloro-2',5'-dihydroxybiphenyl was found in extracts of incubations with 4-chloro-2'- and -3'-monohydroxybiphenyls, while the 4-chloro-3',4'-dihydroxybiphenyl was the only product found from 4-chloro-3'- and -4'-monohydroxybiphenyls. No other chlorinated diols were detected by GC/MS. These data suggest that the major route of biosynthesis of the diols was via a second hydroxylation step and not aromatization of dihydrodiols derived from primary arene oxides. We propose a scheme for the in vitro synthesis of the catechol and hydroquinone metabolites, which may be precursors for electrophilic semiquinone or quinone products with the potential for cytotoxic and genotoxic effects.

Detection of PCB adducts by the 32P-postlabeling technique.

The purpose of this study was to determine whether lower chlorinated biphenyls would be bioactivated to electrophilic metabolites by microsomes alone or in combination with peroxidase. Monochloro- and dichlorobiphenyls were incubated with liver microsomes of rats treated with phenobarbital and beta-naphthoflavone, an NADPH-regenerating system, and deoxyguanosine 3'-monophosphate (dGp). The resultant adducts were analyzed by 32P-postlabeling either following microsomal incubation alone ("preoxidized") or coupled with subsequent oxidation with horseradish peroxidase/H2O ("oxidized"). The incubation of 4-monochlorobiphenyl (4-MCB) resulted in the formation of two minor adducts by microsomal activation alone. However, the oxidized sample showed two additional major adducts. Formation of the latter adducts was almost completely (> 80%) inhibited when the oxidation reaction was performed in the presence of ascorbic acid. The other test mono- and dichlorobiphenyls also formed 1-3 major adducts. Compared with microsomal activation alone, these adducts were enhanced after the oxidation reaction or detected only in the oxidized samples. These data suggest that (1) some adducts of the lower chlorinated biphenyls are derived from arene oxides and (2) many adducts may be formed by metabolism of the parent compounds to catechol and p-hydroquinone species, which are oxidized to semiquinones and/or quinones. The involvement of quinones and/or semiquinones was supported by UV/vis spectroscopic measurements, which showed that metabolites of 4-MCB can be oxidized to products with spectra characteristic of quinones. These data raise the possibility that lower chlorinated biphenyls may be genotoxic and may explain the fact that commercial polychlorinated biphenyl mixtures are complete rodent carcinogens.

Possible etiologic heterogeneity of vulvar intraepithelial neoplasia. A correlation of pathologic characteristics with human papillomavirus detection by in situ hybridization and polymerase chain reaction.

A correlated histopathologic and molecular virologic study of 30 cases of vulvar intraepithelial neoplasia Grade 3 (VIN 3) and six associated invasive vulvar carcinomas was performed. Paraffin sections were examined for human papillomavirus (HPV) types 6, 11, 16, and 18 by in situ hybridization for viral transcripts and by polymerase chain reaction (PCR) for amplification of HPV and of the beta-globin gene. Vulvar intraepithelial neoplasia Grade 3 was histologically subclassified into warty (bowenoid) (20 cases) and basaloid (undifferentiated) (ten cases) types. Warty VIN characteristically was composed of squamous cells displaying abnormal proliferation and maturation and an undulating or spiked surface creating a "condylomatous" appearance whereas basaloid VIN had a smooth surface and was composed of undifferentiated basaloid cells resembling carcinoma in situ of the cervix. Human papillomavirus-16 was the only type detected in 16 of 30 VIN 3 and in five of six invasive carcinomas. The HPV-positive women were younger than HPV-negative women (mean age at diagnosis, 49 versus 60 years), their lesions more frequently demonstrated koilocytotic atypia (94% versus 43%), and they were more likely to have warty compared with basaloid VIN lesions (65% versus 30%). These findings suggest that there are at least two different types of VIN which have differing clinical, pathologic, and viral profiles.

Human papillomavirus in women with vulvar intraepithelial neoplasia III.

Untreated cases of vulvar intraepithelial neoplasia (VIN) III may progress to invasive vulvar carcinoma. Tissues from 29 New Zealand women with VIN III were examined for the presence of human papillomavirus (HPV) types 6, 11, 16 and 18 by in situ hybridization and polymerase chain reaction. HPV 16, the only HPV type detected in the lesions, was identified in about half the cases. HPV-positive women were younger than HPV-negative women, and their lesions displayed koilocytosis more often. In four of five cases in which there was a progression to invasive cancer, HPV 16 was detected in both the VIN III and invasive cancer tissue.

Coordinated three-dimensional reconstruction from serial sections at macroscopic and microscopic levels of resolution: the human heart.

This report describes procedures we have developed for obtaining correlated quantitative structural information at two very different levels of resolution. Accurate reconstructions of entire organs and samples of tissue within organs were produced in correct scalar and topographical relationship using computer-assisted techniques. A specially designed sectioning apparatus, a macrovibratome, was used to section serially the ventricles of the human heart macroscopically. Photographs were taken of every slice. A tissue block excised from a slice at a specified locus in the left ventricular wall was embedded in plastic; serial-3 micron sections were cut in each of two orthogonal orientations. Photomicrographs were taken by semi-automated microscopy. Images of both macroscopic and microscopic sections were projected onto a bitpad and manually digitized. The resulting tables of x-, y-, and z-coordinates were reassembled on a VAX 11/750 computer, then transferred to a high-performance graphics workstation and displayed as three-dimensional images. Microscopic images were shown in the correct reference frame with respect to the macroscopic (parent) structure.

Carcinoma in situ of the vulva: a review of 31 treated and five untreated cases.

Thirty six patients with carcinoma in situ of the vulva have been followed from two to 23 years. Among 31 patients managed by surgical excision, there were four recurrences of vulvar carcinoma in situ and one patient developed a vulvar carcinoma 17 years later. Four middle-aged and elderly women managed only by biopsy all progressed to invasive vulvar carcinoma in two to eight years; one additional patient progressed to invasion after inadequate primary treatment. These last five cases all represented multifocal lower genital tract neoplasia. Untreated vulvar carcinoma in situ, when seen as part of a multifocal lower genital tract neoplastic process, in middle and later life is likely to progress to invasion.

Effects of aging at the adrenergic cardiac neuroeffector junction.

Adrenergic neural degeneration was seen to increase with age. This is thought to contribute to the decreased cardiac content of the transmitter. Pharmacologically, it was found with the use of tyramine that virtually all of the norepinephrine (NE) pool is available for release, and that there is no difference in the amount of NE released in relation to age. Cardiac responsiveness to adrenergic agonists decreases with age. Our results suggest that this is caused in great measure by increased activity of the prejunctional, neuronal uptake mechanism in the older animal.

Fractures of the lateral process of the talus.

A retrospective review of nine patients with fractures of the lateral process of the talus indicates that a substantial portion of patients will have persistent symptoms if the fracture is not diagnosed and appropriately treated soon after the injury. When a patient is evaluated for the symptoms of a "sprained ankle," these fractures are often overlooked on the initial roentgenograms. If untreated, these fractures often fail to heal, and persistent pain over the lateral aspect of the ankle following an inversion injury should be investigated for the possibility of this diagnosis. Prompt treatment of acute fractures appears to lead to the best result. Nondisplaced fractures heal well in a short-leg cast, with six weeks of immobilization. Large displaced fracture fragments require surgical treatment: single large fragments should be reduced and internally fixed, and large comminuted fragments should be excised.

The invasive potential of carcinoma in situ of the cervix.

Nine hundred and forty-eight patients with carcinoma in situ (CIS) of the cervix diagnosed histologically have been followed from five to 28 years. Among the 817 patients who had normal cytology follow-up, 12 (1.5%) developed invasive carcinoma. A second group of 131 patients continued to produce abnormal cytology consistent with cervical neoplasia, and 29 (22%) of them developed invasive carcinoma of the cervix or vaginal vault. Patients with continuing abnormal cytology after initial management of CIS of the cervix are 24.8 times more likely to develop invasive carcinoma than women who have normal follow-up cytology. Further, when compared with the population at large, the chances of patients with normal follow-up cytology developing invasive cervical or vaginal vault carcinoma increase 3.2-fold over women who have never had CIS of the cervix.

An ultrastructural study of the effects of age on sympathetic innervation and atrial tissue in the rat.

Previous work on the rat heart has demonstrated an age-related reduction in catecholamines and a decline in myocardial cell sensitivity to catecholamines in vitro. We used ultrastructural cytochemical techniques to label noradrenergic vesicles of the sympathetic nerve terminals of the rat heart atrium, and addressed the question of whether these deficits are accompanied by a decrease in the number of synaptic vesicles or by progressive axonal degeneration. Our results demonstrate a significant sympathetic axonal degeneration between 3 and 24 months of age. No decrease in noradrenergic vesicle population in the intact nerve terminals could be discerned over this age span. Atrial cell structural alterations observed with age include: (1) increased quantities of residual bodies; (2) infrequent but definite myofibrillar disorganization at cell peripheries; (3) infrequent regional discontinuity of cell attachments and (4) increased extracellular collagen. We suggest that the apparent integrity of noradrenergic vesicle populations is consistent with reports by other investigators that levels of the catecholamine synthesizing enzyme, tyrosine hydroxylase, in sympathetic ganglia increase with age. The previously observed decline in cardiac catecholamines with age may be due to axonal degeneration rather than to reduced noradrenergic vesicles in intact terminals.

The effect of bovine lung heparin on normal human platelets as measured by electronic particle size analysis.

The response of normal human platelets to treatment with bovine lung heparin was evaluated using electronic particle size analysis and aggregometry. Samples for electronic particle size analysis were obtained both before and after the addition of heparin to platelet-rich plasma (PRP). The number of single platelets decreased significantly after the addition of heparin to PRP obtained from 22 individuals. This decrease averaged 18% in 19 samples which did not show a significant increase in transmittance and was at least 70% in 3 samples which showed a significant increase in transmittance. The size distribution of single platelets was not significantly altered. In addition, electron microscopic examination revealed that platelets treated with heparin are activated.

Collagen-induced platelet aggregation as studies by electronic particle size analysis.

The collagen-induced response of free platelets during aggregation was measured with an electronic particle size analyzer. Aggregation was induced in platelet-rich plasma by 0.05 mg/mL collagen. The reaction was followed turbidimetrically and samples for electronic particle size analysis were obtained during aggregation. Free platelet number decreased rapidly after the addition of collagen. This decrease preceded detectable changes in transmittance and a significant reduction in free platelet number was measured during the classical lag phase. In addition, the size distribution of free platelets at various times after the addition of collagen was not significantly altered indicating that large platelets are not preferentially involved in collagen-induced aggregation.

Heparin induced platelet aggregation: in vitro confirmation of thrombotic complications associated with heparin therapy.

Eleven patients who developed thromboembolic complications while receiving heparin were studied for a possible adverse reaction to heparin as the cause of their progressive thrombosis. Fifteen additional patients who were receiving heparin for recurrent thromboembolism, but who did not develop signs of thrombotic complications, were studied as patient controls. The most significant finding was an abnormal in vitro aggregation response to heparin alone in all of the patients who developed complications who were tested for it (64 percent). None of the patient controls demonstrated this abnormality. In addition, thrombocytopenia was noted in all of the former but in only one of the latter. Results of prothrombin times, fibrinogens and fibrin split products eliminated disseminated intravascular coagulation as the cause of the thrombocytopenia in the majority of cases. Finally, an approach to the early detection of the abnormal heparin response is presented and guidelines for its therapeutic management are recommended.