Effects of vitamin D3 supplementation and UVb exposure on the growth and plasma concentration of vitamin D3 metabolites in juvenile bearded dragons (Pogona vitticeps).

The effectiveness of dietary vitamin D3 and UVb exposure on plasma vitamin D metabolites in growing bearded dragons (Pogona vitticeps) was studied. A total of 84 (40 males and 44 females) newly hatched bearded dragons were allocated to six levels of oral vitamin D3 supplementation (0 to 400%) or six UVb exposure times (2 to 12 h). At 3 and 6 months of age, blood samples were obtained from each animal and analysed for 25(OH)D3 and 1,25(OH)2D3. At 3 months of age, plasma concentrations of 25(OH)D3 did not increase with increasing vitamin D3 supplementation unlike the 1,25(OH)2D3. At 6 months of age, plasma concentrations of both 25(OH)D(3) and 1,25(OH)2D3 increased with increasing vitamin D(3) supplementation. Plasma concentrations in UVb-exposed animals were 18 times higher for 25(OH)D3 (178.4+/-9.0 vs. 9.9+/-1.3 nmol/L) and 5.3 times higher for 1,25(OH)2D3 (1.205+/-0.100 vs. 0.229+/-0.025 nmol/L) than in vitamin D(3) supplemented animals at 6 months of age. This study shows that 2h of UVb exposure enables adequate physiological concentrations of plasma vitamin D metabolites to be maintained in growing bearded dragons. Oral supplementation of vitamin D(3) is ineffective in raising plasma concentrations of 25(OH)D3 and 1,25(OH)2D3 to concentrations observed in UVb-exposed animals.

Evidence for early local viral replication and local production of antiviral immunity upon mucosal simian-human immunodeficiency virus SHIV(89.6) infection in Macaca nemestrina.

Transmission of human immunodeficiency virus type 1 (HIV-1) is largely a result of heterosexual exposure, leading many investigators to evaluate mucosal vaccines for protection against intravaginal (i.vag.) transmission in macaque models of AIDS. Relatively little is known, however, about the dynamics of viral replication and the ensuing immune response following mucosal infection. We have utilized a simian-human immunodeficiency virus (SHIV) to study the differences in viremia, CD4 T-cell percentages, and mucosal and systemic anti-SHIV humoral and cellular immune responses during primary infection of animals infected either intravenously (i.v.) or i.vag. Positive viral cocultures, peripheral blood mononuclear cell viral load peaks, and CD4 cell declines were delayed by 1 week in the i.vag. inoculated animals compared to the animals infected i.v., demonstrating delayed viral spreading to the periphery. In contrast, mucosal anti-SHIV antibody levels were greater in magnitude and arose more rapidly and mucosal CD8(+) T-cell responses were enhanced in the i.vag. group animals, whereas both the magnitudes and times of onset of systemic immune responses for the animals in the two groups did not differ. These observations demonstrate that compartmentalization of viral replication and induction of local antiviral immunity occur in the genital tract early after i.vag. but not i.v. inoculation. Induction of mucosal immunity to target this local, contained replication should be a goal in HIV vaccine development.

Adenosine kinase of Arabidopsis. Kinetic properties and gene expression.

To assess the functional significance of adenosine salvage in plants, the cDNAs and genes encoding two isoforms of adenosine kinase (ADK) were isolated from Arabidopsis. The ADK1- and ADK2-coding sequences are very similar, sharing 92% and 89% amino acid and nucleotide identity, respectively. Each cDNA was overexpressed in Escherichia coli, and the catalytic activity of each isoform was determined. Both ADKs had similar catalytic properties with a K(m) and V(max)/K(m) for adenosine of 0.3 to 0.5 microM and 5.4 to 22 L min(-1) mg(-1) protein, respectively. The K(m) and V(max)/K(m) for the cytokinin riboside N(6)(isopentenyl) adenosine are 3 to 5 microM and 0.021 to 0.14 L min(-1) mg(-1) protein, respectively, suggesting that adenosine is the preferred substrate for both ADK isoforms. In Arabidopsis plants, both ADK genes are expressed constitutively, with the highest steady-state mRNA levels being found in stem and root. ADK1 transcript levels were generally higher than those of ADK2. ADK enzyme activity reflected relative ADK protein levels seen in immunoblots for leaves, flowers, and stems but only poorly so for roots, siliques, and dry seeds. The catalytic properties, tissue accumulation, and expression levels of these ADKs suggest that they play a key metabolic role in the salvage synthesis of adenylates and methyl recycling in Arabidopsis. They may also contribute to cytokinin interconversion.

Phenotypic instability and rapid gene silencing in newly formed arabidopsis allotetraploids.

Allopolyploid hybridization serves as a major pathway for plant evolution, but in its early stages it is associated with phenotypic and genomic instabilities that are poorly understood. We have investigated allopolyploidization between Arabidopsis thaliana (2n = 2x = 10; n, gametic chromosome number; x, haploid chromosome number) and Cardaminopsis arenosa (2n = 4x = 32). The variable phenotype of the allotetraploids could not be explained by cytological abnormalities. However, we found suppression of 20 of the 700 genes examined by amplified fragment length polymorphism of cDNA. Independent reverse transcription-polymerase chain reaction analyses of 10 of these 20 genes confirmed silencing in three of them, suggesting that approximately 0.4% of the genes in the allotetraploids are silenced. These three silenced genes were characterized. One, called K7, is repeated and similar to transposons. Another is RAP2.1, a member of the large APETALA2 (AP2) gene family, and has a repeated element upstream of its 5' end. The last, L6, is an unknown gene close to ALCOHOL DEHYDROGENASE on chromosome 1. CNG DNA methylation of K7 was less in the allotetraploids than in the parents, and the element varied in copy number. That K7 could be reactivated suggests epigenetic regulation. L6 was methylated in the C. arenosa genome. The present evidence that gene silencing accompanies allopolyploidization opens new avenues to this area of research.

Agency for Toxic Substances and Disease Registry's 1997 priority list of hazardous substances. Latent effects--carcinogenesis, neurotoxicology, and developmental deficits in humans and animals.

In support of Superfund re-authorization legislation, the Division of Toxicology of the Agency for Toxic Substances and Disease Registry (ATSDR) prepared a chemical-specific consultation document for Congress that identified those chemicals with carcinogenic, neurological, or developmental adverse effects having a latency period longer than 6 years. The review was limited to the top 50 substances listed on ATSDR's 1997 Priority List of Hazardous Substances (Priority List). Among the top 50 chemicals, a review of the technical literature indicated that 38 (76%) were classified as "reasonably anticipated," "possibly," or "probably" capable of causing cancer in humans, based either on human and animal data. Eight chemicals (16%) had well-established cancer latency periods in humans of 6 years or more following exposure. Three substances (6%)--arsenic, creosote, and benzidine--had data indicating latency periods longer than 6 years. The technical literature review likewise confirmed the potential for neurological and developmental effects with a latency of 6 years. Twenty-seven (54%) of the top 50 substances caused acute and/or chronic neurotoxic effects; a number of these also caused neurological effects that persisted beyond 6 years (or the equivalent in animal studies) such as: behavioral problems, neurological deficiencies, reduced psychomotor development, cognitive deficiencies, and reduced IQ. Twenty-eight substances (56%) caused adverse developmental effects in offspring of exposed individuals or animals including increased fetal and infant mortality, decreased birth weights and litter sizes, and growth delays. Latency periods for related chemicals are expected to be similar due to structural and toxicological similarities.

The Agency for Toxic Substances and Disease Registry's role in development and application of biomarkers in public health practice.

An overview of the Agency for Toxic Substances and Disease Registry's (ATSDR) biomarker program is presented in the context of the paradigm for biomarkers developed by the National Research Council (NRC, 1987, 1991). The status and projected utility of four biomarker studies conducted by NRC and sponsored by ATSDR, the Environmental Protection Agency (EPA), and the National Institute of Environmental Health Sciences (NIEHS) are discussed. These studies include a review of relevant research on biomarkers for specific toxicologic end points, including reproductive toxicology, pulmonary toxicology, neurotoxicology, and immunotoxicology. Also, the scope of related research on exposure characterization being conducted by the ATSDR-sponsored research program at Rutgers University is reviewed. The potential impact of biomarkers on public health assessments and on the range of ATSDR programs is described. Specifically, the role of biomarkers in dose reconstruction, in ATSDR's health studies program, and in the emerging field of molecular epidemiology is reviewed. In addition, future directions and research needs are addressed.

Cancer policy framework for: public health assessment of carcinogens in the environment.

Cancer remains at the forefront of public health concerns in the United States and throughout the world. Over the past 20 years a wide range of federal agencies and other organizations have been involved in developing policy statements, classification strategies, and assessment methods to address carcinogenesis and health risks. Each of these documents was developed in response to issues confronted by those organizations in pursuing their mission, often as a direct function of legislative mandates. In pursuing its mandated responsibilities, the Agency for Toxic Substances and Disease Registry (ATSDR) must address public health concerns associated with exposure to carcinogens in the context of all available relevant information. This information includes both technical data as well as science policy positions adopted by the range of organizations with programs germane to the assessment and/or regulation of carcinogens. Because of distinct differences in perspective, practice, and policy dictated by the mandated activities of these organizations and the rapidly evolving understanding of carcinogenesis, apparently divergent positions may be reflected in their conclusions. The differences outlined above, coupled with requests from the public, other agencies, and the private sector for a statement reflecting the Agency's position on science and science policy issues related to cancer, prompted the development of this policy. This document is intended to serve as a framework to guide the Agency in its programs and actions regarding carcinogens and to harmonize such efforts with those of other federal agencies and relevant organizations. This framework reflects an assessment of current practice within the Agency and defines the appropriate roles of conclusions derived by other groups, professional judgment, and emerging scientific principles in ATSDR's public health assessments of exposures to carcinogens. This Cancer Policy Framework is not intended to encompass the development of operational guidelines per se, although the Agency recognizes the utility of such efforts. A central theme of this Cancer Policy Framework is the use of risk analysis as an organizing construct based on sound biomedical and other scientific judgment to define plausible exposure ranges of concern rather than single numerical conclusions that may convey an artificial sense of precision. The development and use of innovative tools for exposure and dose response assessment (with particular emphasis on molecular epidemiology) are also endorsed.

In vitro differential metabolism of merbarone by xanthine oxidase and microsomal flavoenzymes. The role of reactive oxygen species.

Merbarone (MB), a nonsedating derivative of thiobarbituric acid, was recently found to induce profound hypouricemia. When incubated with xanthine oxidase (XO) and hypoxanthine in vitro, MB is both an inhibitor of XO and degraded by the XO-hypoxanthine interaction. Compared with allopurinol (Ki = 0.025 microM), MB is a very weak inhibitor of XO (Ki = 51 +/- 8 microM). MB interacts with XO in the presence of hypoxanthine to yield three chromatographically separate products. One of these products has been identified by HPLC retention time and spectral characteristics as 2-oxo-2-desthiomerbarone (2-oxo-MB). The other two products are thought to be S-oxide intermediates in the oxidative desulfuration of this drug. Formation of these products was blocked by catalase, suggesting that the conversion was dependent on reactive oxygen species (especially H2O2) generated by the hypoxanthine-XO system. This suggestion was confirmed by incubating MB with H2O2. In vitro studies with rat liver microsomes have documented the formation of 2-oxo-MB and 4'-OH-MB (4'-OH-MB), the latter being identified by the characteristic HPLC retention time of its acetylated derivative. The formation of 4'-OH-MB has many characteristics of a cytochrome P-450-dependent monooxygenase reaction (NADPH requirement and SKF 525-A inhibition); formation of 2-oxo-MB occurs by a different mechanism that is, as yet, uncharacterized. Incubation of kidney microsomes with MB generated 2-oxo-desthiomerbarone but no detectable 4'-OH-MB.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I clinical and pharmacological study of merbarone.

Merbarone, a nonsedating derivative of thiobarbituric acid, has demonstrated excellent activity against certain murine tumors, including L1210 and P388 leukemias, B16 melanoma, and M5076 sarcoma. Preclinical studies suggested that the antitumor effects of this drug were schedule dependent, since repeated dosing increased killing of tumor cells when compared to intermittent injections. We have completed a Phase I clinical and pharmacological study of merbarone in which the drug was administered both as a 2-h infusion and as a continuous i.v. infusion over 24 h. In view of the increased toxicity observed in animals following bolus injections and the possibility of schedule-dependent anticancer activity, a schedule of drug administration daily for 5 days was selected. Fifty patients with advanced cancer were treated at dose levels that ranged from 100 to 1500 mg/m2/day. When the drug was administered by peripheral vein, phlebitis was observed at the infusion site at daily doses greater than or equal to 150 mg/m2. Therefore, all patients who received drug doses greater than or equal to 200 mg/m2 were treated by continuous i.v. infusion using central venous catheters. Renal insufficiency, initially observed at a dose of 1000 mg/m2/day, was the dose-limiting toxic reaction at 1500 mg/m2/day. Three of five patients treated at the highest dose level were unable to complete the infusion due to this effect. Marked hypouricemia was observed in all patients. Other toxic effects were mild and included nausea, fatigue, leukopenia, thrombocytopenia, and anorexia. Alopecia was noted in several patients who received doses greater than or equal to 1000 mg/m2/day. No major antitumor effects were observed. Dose-dependent, steady-state plasma concentrations of merbarone were reached within 24-48 h after beginning the continuous i.v. infusion. Elimination of drug from plasma followed a two-compartment model, with a t1/2 alpha of 4.2 h and a t1/2 beta of 15.3 h. Renal excretion of merbarone and its major metabolites accounted for less than 30% of the administered dose. We conclude that merbarone is relatively well tolerated with few constitutional symptoms. The current formulation of the drug causes phlebitis when administered by peripheral vein, and renal insufficiency is commonly observed at daily doses which exceed 1250 mg/m2. The recommended dose for extended Phase II evaluation is 1000 mg/m2/day daily for 5 days administered by central venous catheter.

Induction of profound hypouricemia by a non-sedating thiobarbiturate.

5-[N-phenylcarboxamido]-2-thiobarbituric acid (merbarone) is a non-sedating derivative of thiobarbituric acid originally developed for anticancer use. In the initial clinical study, a profound reduction in serum uric acid was observed. In 20 patients who received five daily doses of merbarone ranging from 100 to 750 mg/m2, serum uric acid concentration was reduced from a mean pretreatment value of 5.7 +/- 1.6 mg/dL to a mean lowest value of 1.3 +/- 0.5 mg/dL. In most patients, the onset of the effect occurred with 24 hours and was maximal by 48 to 72 hours. Metabolic studies in two patients showed an increase in urinary uric acid excretion within 24 hours after initiation of drug treatment. A marked increase in fractional excretion of uric acid was sustained throughout the period of drug treatment. Urinary excretion of total oxypurines (xanthine and hypoxanthine) was increased twofold to threefold relative to baseline levels. Ultrafiltration studies showed that merbarone did not significantly displace binding of urate from albumin. When merbarone was incubated with xanthine oxidase in vitro, several reaction products were observed, including 2-oxo-2-desthio-merbarone and a compound with retention time similar to 4'-OH-merbarone. Both of these compounds have been described previously as metabolites of merbarone in human subjects. The parent drug and both metabolites were found to inhibit xanthine oxidase (Ki = 41, 36, and 240 mumols/L, respectively). However, this inhibitory effect was substantially less potent than allopurinol (Ki = 0.025 mumols/L). This study indicates that merbarone induces profound hypouricemia primarily by increasing uric acid excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

4-demethoxydaunorubicin (idarubicin) in combination with 1-beta-D-arabinofuranosylcytosine in the treatment of relapsed or refractory acute leukemia.

We conducted a Phase I-II trial of 4-demethoxydaunorubicin (idarubicin, IDR) in combination with 1-beta-D-arabinofuranosylcytosine (ara-C) in 51 patients with relapsed or refractory acute nonlymphocytic leukemia, acute lymphocytic leukemia, or chronic myelogenous leukemia in blast crisis. Only 1 of 12 patients treated at the first dose level (idarubicin, 10 mg/m2/day for 3 days and ara-C, 25 mg/m2 i.v. bolus followed by 200 mg/m2 continuous infusion daily for 5 days) achieved aplasia and complete remission. The dose of idarubicin was subsequently increased to 10 mg/m2/day for 4 days with the ara-C dose held constant. Complete remission incidence for this dose schedule was: 7 of 31 patients with acute nonlymphocytic leukemia, 0 of 5 patients with acute lymphocytic leukemia, 0 of 1 patient with chronic myelogenous leukemia in blast crisis, and 1 of 2 patients with biphenotypic leukemia. Nonhematological toxicity included nausea, vomiting, mucositis, and abnormal liver function tests. Detailed pharmacological studies were performed to determine whether ara-C altered IDR metabolism or that of its main metabolite, 13-hydroxyidarubicinol or IDR clearance. A high degree of variability among patients was apparent and no consistent effect could be demonstrated. In summary, 9 of 37 patients (24%) with relapsed or refractory ANLL, including 1 patient with biphenotypic leukemia, achieved remission. We conclude that idarubicin in combination with ara-C is an active combination in patients with relapsed or refractory leukemia.

Phase I trial and clinical pharmacological evaluation of hexamethylene bisacetamide administration by ten-day continuous intravenous infusion at twenty-eight-day intervals.

We have treated 33 patients with different types of advanced cancer by 10-day continuous i.v. infusion courses of hexamethylene bisacetamide (HMBA), a drug that produces differentiation of a variety of transformed cell lines on prolonged exposure in vitro to drug concentrations of 3 to 5 mM. In this dose-finding and pharmacokinetic study, five dosage levels were explored from 12 to 28 g/m2/day. Patients who had not shown progression of disease were given repeat courses of therapy at 28-day intervals. Seventy-two courses of therapy were administered; 17 patients received one course; eight patients received two; six patients received three; and one patient each received four and 17+ courses, respectively. The maximal tolerated dose was 28 g/m2/day for 10 days; the dose-limiting toxic effects were thrombocytopenia with hemorrhage and central nervous system dysfunction manifesting as disorientation and confusion. Based on these studies the recommended dosage for Phase II studies by the 10-day schedule is 24 g/m2/day. Pharmacokinetic studies demonstrated rapid clearance of HMBA from plasma; the decay phase data fit a one compartment model with a mean plasma half-life of 2.5 h and a range from 0.6 to 5.8 h. Mean plasma steady-state levels in our patients were 0.37, 0.58, 0.86, 0.88, and 1.42 mM, at the 12-, 16-, 20-, 24-, and 28-g/m2/day dosage levels, respectively. The data indicate that plasma HMBA concentrations of 1 mM can be maintained for 10 days with acceptable patient tolerance, but that HMBA concentrations in excess of 1.4 mM for 10 days are associated with substantial hematological and central nervous system toxicity. Objective antitumor effects were observed in five patients; one woman with non-small cell lung cancer, who has received 17+ courses over a period of 28+ mo, achieved a partial remission that continues at 28+ mo on therapy. Transient regression of cutaneous metastases was observed in three patients with breast carcinoma and one patient with colorectal carcinoma.

"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple forms and fragments of cytosolic glucocorticoid receptors from human leukemic cells and normal lymphocytes.

Therapy with glucocorticoids is generally more effective in acute lymphoblastic leukemia than in other types of human leukemia. Previous studies, however, have not revealed any consistent relationship between clinical responsiveness and the cellular or cytosolic concentration of glucocorticoid-binding sites. The objectives of this study were to determine whether there are intrinsic structural differences among the glucocorticoid receptors in various types of leukemic cells and normal lymphocytes and to investigate the role of endogenous peptidases in receptor degradation. Cytosols were prepared from fresh or rapidly frozen leukocytes from 6 healthy adults and 35 high-risk leukemia patients (median white blood cell count, 150,000 cells/microliter; median age, 13 years). Receptors were labeled with [3H]triamcinolone acetonide and quantitated by charcoal-dextran treatment or Sephadex LH-20 chromatography. Mean and median cytosolic receptor concentrations in 12 acute lymphoblastic leukemia specimens lacking the standard B-cell or T-cell markers ("null cells") were approximately 4-fold higher than in 23 other leukemic cell specimens. No other consistent differences in receptor content were observed. Agarose filtration and ultracentrifugation in hypotonic buffers containing 20 mM Na2MoO4 revealed complexes of similar size and shape in all clinical specimens tested and two established leukemic cell lines. They had Stokes radii (Rs) of 8.1 +/- 0.5 (S.D.) nm (n = 50), sedimentation coefficients of 9.5 +/- 0.3S (n = 40), molecular weights of approximately 330,000, and axial ratios (a/b) of approximately 12. In hypertonic, molybdate-free buffer, these oligomeric complexes were dissociated into subunits with Rs of 5.9 +/- 0.3 nm (n = 12) and a/b of 11 to 12, as observed previously for other receptors. Fragmentation of the oligomer and the subunit was evident in some cytosols. High activities of peptidases of various specificities were detected in leukemic cell cytosols, as in other cytosols, by fluorometric assays with derivatives of 7-amino-4-methylcoumarin. Receptor cleavage by these and other endogenous enzymes may account for previous observations of "abnormal" receptors in cytosols from some leukemic specimens. We conclude that intrinsic structural defects in the receptors are unlikely explanations for the unresponsiveness of some types of leukemia to steroid therapy.

Structure, dissociation, and proteolysis of mammalian steroid receptors. Multiplicity of glucocorticoid receptor forms and proteolytic enzymes in rat liver and kidney cytosols.

The forms of steroid receptors detected in mammalian tissue cytosols vary from a globular fragment, the mero-receptor, with a molecular weight (Mr) of approximately 23,000 to highly asymmetric molybdate-stabilized complexes (Mr approximately 330,000). Our objectives were to investigate the relationships among various receptor forms and mechanisms of stabilization by Na2MoO4, to characterize endogenous proteolytic enzymes, and to evaluate the effects of freezing the tissues on receptor structure and protease activities in the resultant cytosols. Glucocorticoid receptors and proteases were analyzed in cytosols from fresh and frozen rat liver and kidney. Enzymes were assayed fluorometrically at approximately 24 degrees C, with peptidyl tyrosine, lysine, and phenylalanine derivatives of 7-amino-4-methylcoumarin. Rates of cleavage of the tyrosine-containing substrate by crude and partially purified liver cytosol enzymes were markedly suppressed by Na2MoO4. While receptor cleavage by these enzymes has not been demonstrated, these results illustrate the direct inhibition of proteolysis by molybdate. Rates of cleavage of the lysine-containing substrate were 7- to 40-fold higher in cytosols from frozen liver and fresh or frozen kidney, in which the Stokes radius (Rs) of the largest receptor form was 70-74 A, than in fresh liver cytosol, in which Rs was 84 +/- 2 A (n = 20). Filtration of the 84 A complex (Mr approximately 330,000) in hypertonic buffer without Na2MoO4 revealed a 7:3 mixture of forms with Rs of 50-60 A (Mr approximately 90,000) and Rs of 30-40 A (Mr approximately 50,000). The latter are derived from the 50-60 A forms by proteolysis and/or dissociation. We conclude that the 84 A (untransformed) receptor is an oligomer, probably a tetramer, containing the 50-60 A subunits and that purification of the intact structure will require removal or inactivation of contaminating proteases.

Absence in glucocorticoid-resistant mouse lymphoma P1798 of a glucocorticoid receptor domain responsible for biological effects.

Glucocorticoid-resistant (CR), in contrast to glucocorticoid-sensitive (CS), mouse lymphoma P1798 was shown to lack antiglucocorticoid receptor immunoactivity. Antibodies raised against the purified rat liver glucocorticoid receptor (GR) cross-reacted with the GR from CS, but not with the GR from CR, P1798 lymphoma. Using highly specific antisera against the GR in an indirect competitive enzyme-linked immunosorbent assay, it was demonstrated that alpha-chymotrypsin digestion of the GR from CS P1798 lymphoma caused a separation of a "resistant-like" nonimmunogenic steroid and DNA-binding domain (Stokes' radius, 3.3 nm) from an immunoactive domain (Stokes' radius, 2.6 nm). In contrast to CS P1798 lymphoma, neither before nor after alpha-chymotrypsin digestion, immunoactivity could be found in the cytosol from CR P1798 lymphoma. This was assayed after chromatography on DNA-cellulose or gel filtration on Agarose A (0.5 m). These results suggest that the domain of the CS GR containing the immunoactive determinant(s), normally removed by limited proteolysis by alpha-chymotrypsin, appears to be missing in CR P1798 lymphoma cytosol. It seems that this domain plays an important role in the mechanism of action of glucocorticoids. This might suggest that a mutation has occurred affecting the genome resulting in defective transcription of the receptor gene(s) in CR P1798 lymphoma.

Rural women in Africa and technological change: some issues.

PIP: The attempt is made in this discussion to highlight some of the important sociological and technical issues relating to rural women in Africa and technological change which appear to have been underplayed, misconceived or overlooked in the past. Attention is directed to the rural woman as a member of the family unit, the image of the rural man, rural women as a diversified group, community and national governmental commitment to rural technology innovations, the use of already existing traditional groups and institutions to effect rural technological change, and design specifications and shortcomings of equipment and tools (manufacturing costs, exploitation of locally available energy resources, the simplicity of the devices), and infrastructural and marketing problems. Numberous projects aimed at improving the lot of women in the rural areas have focused only on women, rather than the woman as a member of an extended as well as a nuclear family unit. Consequently, they have failed, for rural women do not exist or operate in isolation. It is difficult to believe the overall image in much of the literature that the husbands of rural women show no sympathy or regard for their wives. In the effort to attract investment to improve upon the position of rural women, reality should not be distorted with this one-sided view. Men should be involved in the technology planned for rural women, and the technological change should be planned and implemented in such a way that it results in an improvement in the relationship between the rural couple and generally between members of the rural family and between males and females in the village. Another problem is overgeneralization, and it must be recognized that considerable differentiation exists between rural women themselves. The importance of community, governmental and political commitment to rural technology innovations in order to ensure their success is neglected in the literature. The government and polictical leadership can do much to introduce improved technologies in the rural areas. The use of existing traditional institutions to bring about tecnological change in the rural areas needs to be stressed. Primary reasons why some of the improved devices introduced for use by rural women have been rejected include the following: the devices fail to meet the priority needs of the women and socioeconomic and cultural factors are not considered in their design. Most developing countries are without the required industries to produce the needed basic components. Exploitation of some of the available natural resources would make life much easier for rural women. As rural societies are usually imperfectly linked to bacly organized markets, infrastructural facilities, such as feeder roads, would have to be improved. The following are among the hypotheses suggested by this review: technological innovations linked to existing traditional skills and methods are likely to have easier acceptance in the rural areas than those divorced from these skills and methods; and technological innovations which are disseminated through existing traditional institutions and groups are likely to have easier acceptance. Guidelines for future research are included.^ieng