Obtaining a High Percentage of Explants with Negative Serological Reactions against viruses by Combining Potato Meristem Culture with Antiphytoviral Chemotherapy

A high percentage of plantlets, in which PVX, PVM, and PVS were no longer detectable by precipitin tests was obtained from 100% virus-infected stocks of the potato varieties Rosa and Priobskij rannija by combining meristem (tip) culture with antiphytoviral chemotherapy. When 2,4 - dioxohexahydro 1,3,5-triazinc (DHT) was added to the nutrient media, no virus was detectable serologically in 72% of the corresponding explants of the variety Rosa and in 66% of that of the variety Priobskij rannija, respectively. Contrary to this, all untreated control explants proved virusinfected. Treatment with 2-thiouracil resulted in 46.6%, with cyanoguanidine in 28.6% of plantlets, in which no virus was detectable serologically.     

百合不定芽培养脱毒种球生产的研究

百合不定芽培养再生植株的病毒脱毒效果因百合品种和病毒种类而有所差异;‘卡萨布兰卡’百合潜在病毒（LSV）容易脱去,脱毒率达76%,‘魅丽’黄瓜花叶病毒（CMV）能全部脱去。‘卡萨布兰卡’不定芽培养在固体培养基和液体培养基都能很好形成子球;在含有抗病毒剂（10 mg/L DHT）的液体培养基中子球增大显著,LSV脱毒效果理想。无病毒子球移到有防虫网的户外栽培,2年后部分植株被检测出病毒,再感染率因品种而不同,‘卡萨布兰卡’高达73%,麝香百合‘乔治亚’仅17%;无病毒植株‘乔治亚’,香华丽百合和‘卡萨布兰卡’的生长高度要比有病毒植株明显增高。     

Freeing Sweet and Sour Chery Trees from Prunus Necrotic Ringspot Virus by Injections of 2,4-Dioxohexahydro-1,3,5,-triazine (DHT)

After three injections of 1 ml of 0.01% DHT solutions under the cortex of the stems of 100% artificially or naturally with prunus necrotic ringspot virus infected sweet and sour cherry plants of 4 varieties axillary buds were grafted onto the indicator plant Shirofugen. 99% of these buds proved virus free. Contrary to this, only 28 to 32% of the buds from solvent injected control plants proved free of viruses. About 65% of the fruit-trees grown up from buds of DHT treated plants were still virus free, when the test procedures were performed again two years later. Treatments of the sour cherry variety Podbelskaja with DHT were by far less effective.     

Adrenal and gonadal steroids inhibit IL-6 secretion by human marrow cells

Adrenal and gonadal steroids have protective effects on the skeleton that may be conferred partly by their ability to inhibit bone resorptive cytokines such as interleukin 6 (IL-6). We tested the hypothesis that IL-6 secretion by human marrow cells and a line of marrow stromal cells (KM101) is inhibited by dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and 17beta-oestradiol (E(2)). We also examined whether the estrogen status of the donor influenced the steroids' effects on IL-6 secretion. Femoral bone marrow was obtained from 19 postmenopausal women undergoing hip arthroplasty, and from seven subjects receiving oestrogen replacement therapy (ERT) at the time of surgery. Low-density mononuclear cells were isolated and cultured in IL-1beta-supplemented media, with or without DHEA, DHT or E(2). DHEA suppressed IL-6 more consistently than DHT or E(2): DHEA significantly suppressed IL-6 in 84% of cultures, DHT suppressed IL-6 in 58%, and E(2)did so in 50%. The magnitude of IL-6 inhibition was also greater for DHEA (group mean, treated/control of 62%) compared to DHT (81%) and E(2)(76%). In cultures from subjects receiving ERT, DHEA and DHT suppressed IL-6 in some, whereas E(2)did not suppress IL-6 secretion. Each steroid also significantly inhibited IL-6 secretion by KM101 cells. In summary, in marrow cultured from postmenopausal women, DHEA suppressed IL-6 secretion more consistently and to a greater degree than did DHT and E(2). Second, the inhibitory effect of E(2)was abrogated in marrow from women receiving ERT.     

Testosterone regulation of sex differences in fetal lung development.

Fetal lung development, in particular surfactant synthesis, exhibits a sexual dimorphism. Dihydrotestosterone (DHT) has been shown to delay fetal pulmonary surfactant production, but the potential role for testosterone is unknown. Both testosterone and DHT are potent masculinizing hormones, yet in some instances, an end organ specificity for DHT is present. We hypothesized that the delay in fetal lung surfactant production is dependent upon DHT such that inhibition of the synthesis of DHT from the precursor hormone testosterone would eliminate the sex difference by allowing the male fetus to produce surfactant at the female level. We tested this hypothesis using 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5-alpha-androstane-3-one (4-MA), a potent inhibitor of the enzyme 5 alpha-reductase, which converts testosterone into DHT. First, studies were performed in vivo. 4-MA (20 mg/kg/day) or an equivalent volume of vehicle was injected into pregnant rabbits from Day 12 through Day 26 of gestation. On Day 26, the fetuses were delivered, the lungs were lavaged, and fetal sex was noted. Treatment with 4-MA resulted in a lack of any male-female difference in the anogenital distance and no DHT was detected in the serum of any treated fetus. Phosphatidylcholine (PC), saturated phosphatidylcholine (SPC), and sphingomyelin (S) were measured in the lung lavage, and were expressed as the ratios of PC to sphingomyelin (PC:S) and SPC to sphingomyelin (SPC:S). Sex differences in the PC to sphingomyelin ratio of 4-MA-treated fetuses (female PC:S ratio, 1.43 +/- 0.14; male PC:S ratio, 1.00 +/- 0.13 [mean +/- SE]; P = 0.04) and in the SPC:S ratio of the 4-MA-treated group (female SPC:S ratio, 0.68 +/- 0.10; male SPC:S ratio, 0.35 +/- 0.10; P = 0.03) were present after treatment with 4-MA. The effect of testosterone and of 4-MA on fibroblast pneumonocyte factor (FPF) production was studied in vitro. Fetal rat lung fibroblasts were cultured to confluence with either no added androgen, DHT, testosterone, or testosterone plus 4-MA, and conditioned media for FPF were prepared. Conditioned media were added to fetal Type II cell cultures and FPF activity was measured as the degree of stimulation of the incorporation of [3H] choline into SPC. The conversion of radiolabeled testosterone to DHT by the fibroblasts was inhibited by 4-MA (10(-5) M). Conditioned media from untreated female fibroblasts stimulated with cortisol exhibited significant FPF activity ([3H]choline incorporation into SPC, 140 +/- 17% of control).(ABSTRACT TRUNCATED AT 400 WORDS)     

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.     

Verstärkung der antiphytoviralen Wirkung von 2,4-Dioxohexahydro-l,3,5-triazin durch Kombination mit Verbindungen mit Guanidinstruktur

Intensified antiphytoviral activity of 2,4-dioxohexahydro-l,3,5-triazine by combination with guanidines Combined application of the antiphytoviral compound 2,4-dioxohexahydro-l,3,5 triazine (DHT) and different guanidines (GDs) that were either unsubstituted or substituted only by low-molecular substituents reduced the concentration of potato virus χ (PVX) in leaves or Nicotiana tabacum L. cv. Samsun much more than application of either agent alone. In secondarily PVX-infected leaves, the activity of 2,4-dioxohexahydro- 1,3,5-triazine was increased by a larger number of GDs and to a greater degree than in inoculated ones. The activities of GDs against tobacco mosaic virus (TMV) in Nicotiana tabacum L. cv. Samsun as well as against brome grass mosaic virus (BRV) in Hordeum vulgare L. cv Vogelsanger Gold were only insignificantly increased by combination with DHT. On the other hand, in experiments with so-called identical potato eye cuttings, in which several eye cuttings were obtained from each potato tuber, one serving as a control and the others being treated with DHT, N-cyano guanidine or a combination of these substances, the number of cuttings with symptoms of potato leaf-roll virus (PLRV) could be much more greatly reduced by the combination than by the individual preparations. The number of cuttings with symptoms of potato virus Y and potato virus A was significantly reduced by treatment with the combined preparation, but not by treatment with DHT or N-cyano guanidine alone. Additional investigation with N-cyano-GD, and, beside this, with acetyl-GD and N N′ N triamino-GD indicated a close correlation between the diminution of numbers of potato eye cuttings with virus symptoms and the increase in tuber weight. The greater the reduction in the number of cuttings showing virus symptoms, the greater was the increase in tuber weight. These relationships were observed even in those cases where controls had been treated with ammonium nitrate solutions whose N contents equivalent to the N contents of the preparations. The observed effects of the preparations therefore are not attributable to N-fertilizing effects.     

Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.     

Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.     

Effects of sex hormone binding globulin (SHBG) on human prostatic carcinoma

The purpose of this study was to determine what effects sex hormone binding globulin (SHBG) might have on the growth and steroid content of human prostate carcinoma. Two human prostate carcinoma cell lines were used for this study, ALVA-41 and ALVA-101. The first part of the study was to determine the effect of SHBG or albumin on the uptake of [³H]DHT in the cells. In this experiment both SHBG and albumin inhibits the uptake of [³H]DHT into each of the cell lines when studied in vitro. The degree of inhibition was dependent on the binding capacity of the protein. When [³H]thymidine uptake was measured in each of the cell lines following either the addition of SHBG or albumin to the culture media, an increase in uptake and presumably DNA synthesis was noted in the ALVA-41 and ALVA-101 cells for SHBG additions but not for albumin. Further, this stimulation was increased when testosterone was added to the media, however, [³H]thymidine uptake was decreased by high concentrations of dihydrotestosterone (DHT) or if the SHBG was saturated with DHT prior to being added to the media. The cells also demonstrate high affinity cell membrane receptors for SHBG. Finally, using a 3′, 550 bp cDNA or SHBG, 1.9 and 2.8 kb mRNAs were detected on Northern analysis of the ALVA-101 and ALVA-41 cells. These data indicate SHBG can inhibit uptake of steroids into the prostrate, but also it may act as a stimulus for growth through a SHBG cell surface receptor. In addition, the growth effect may be through an autocrine effect from SHBG or a SHBG-related peptide.     

Nutzung von In-vitro-Pflanzen der Kartoffel zur Prüfung der antiphytoviralen Wirkung von 2,4-Dioxohexahydro-1,3,5-triazin (DHT) in Kombination mit N-Cyanoguanidin

Suitability of in vitro potato plantlets for testing of antiphytoviral effect of a combination of 2,4-dioxo-hexahydro-1,3,5-triazine (DHT) and N-cyano guanidine The suitability of a semiquantitative determination of virus content for the detection of antiphytoviral effects of chemicals was demonstrated by ELISA directly applying at in vitro potato cultures systemically infected and chemotherapeutically treated. The combination of 2, 4-dioxohexahydro-1,3,5-triazine (DHT) and N-cyanoguanidine both applied at a concentration of 0.03% to the culture medium resulted in a high significant reduction of the relative concentration of the potato virus S in the, potato genotype ‘M-812820’ of 44–73 %. A phytotoxic influence of the substances was not observed. The combination of the two antiphytoviral substances had no effect against potato virus Y in explants of the variety ‘Ackersegen’. Differences in the susc, eptibility of various genotypes against biologically active substances were indicated.     

Dihydrotestosterone attenuates hypoxia inducible factor-1α and cyclooxygenase-2 in cerebral arteries during hypoxia or hypoxia with glucose deprivation

Dihydrotestosterone (DHT) attenuates cytokine-induced cyclooxygenase-2 (COX-2) in coronary vascular smooth muscle. Since hypoxia inducible factor-1α (HIF-1α) activation can lead to COX-2 production, this study determined the influence of DHT on HIF-1α and COX-2 following hypoxia or hypoxia with glucose deprivation (HGD) in the cerebral vasculature. COX-2 and HIF-1α levels were assessed via Western blot, and HIF-1α activation was indirectly measured via a DNA binding assay. Experiments were performed using cerebral arteries isolated from castrated male rats treated in vivo with placebo or DHT (18 days) followed by hypoxic exposure ex vivo (1% O(2)), cerebral arteries isolated from castrated male rats treated ex vivo with vehicle or DHT (10 or 100 nM; 18 h) and then exposed to hypoxia ex vivo (1% O(2)), or primary human brain vascular smooth muscle cells treated with DHT (10 nM; 6 h) or vehicle then exposed to hypoxia or HGD. Under normoxic conditions, DHT increased COX-2 (cells 51%; arteries ex vivo 31%; arteries in vivo 161%) but had no effect on HIF-1α. Following hypoxia or HGD, HIF-1α and COX-2 levels were increased; this response was blunted by DHT (cells HGD: -47% COX-2, -34% HIF-1α; cells hypoxia: -29% COX-2, -54% HIF-1α; arteries ex vivo: -37% COX-2; arteries in vivo: -35% COX-2) and not reversed by androgen receptor blockade. Hypoxia-induced HIF-1α DNA-binding was also attenuated by DHT (arteries ex vivo and in vivo: -55%). These results demonstrate that upregulation of COX-2 and HIF-1α in response to hypoxia is suppressed by DHT via an androgen receptor-independent mechanism.     

Effect of DHT (2,4-Dioxohexahydro-1,3,5-triazine) as Well as a Combination of DHT, Alkane Monosulfonate and N-Phenyl-N'-p-carboxyphenyl Thiourea on Tomato Mosaic Virus in Infected Glasshouse Tomato Plants

In two consecutive trials, three treatments of tomato plants of the variety Rivermoon with the antiphytoviral substance DHT (2,4-dioxohexahydro-1,3,5-triazine, 0.15%) 2 d a.i. and 2 and 7 d p.i. reduced the concentration of ToMV (tomato mosaic virus) by 62.3 and 60.5%. The average tomato yields increased by 40 and 26 % compared with the virus diseased, untreated controls, but the yields of the healthy controls were not achieved. A combined treatment of DHT, alkane monosulphonate and N-phenyl-N'-p-carboxyphenyl thiourea only resulted in a minor reduction in the ToMV titre and only a slight increase in yield, compared with the DHT-solo treatment.     

Conversion of dihydrotestosterone to androstanediol glucuronide by female sexual skin

Sexual skin biopsies from 13 normal women were obtained and minces/3-h studied after adding either [3H]dihydrotestosterone (DHT) or [3H]androstanediol (3 alpha diol) to RPMI-1640 medium in a Dubnoff apparatus. Unconjugated or conjugated androgens (after hydrolysis) were purified by three chromatography steps. Formation of 3 alpha diol and 3 alpha diol glucuronide (3 alpha diolG) was linear with time. The conversion of DHT to DHT17 beta G was only 4.4 +/- 0.5%/200 mg/3 h, while conversion to 3 alpha diol was 32 +/- 1.7%. The back conversion of 3 alpha diol to DHT was 30 +/- 3% and conversion to 3 alpha diolG was 4.5 +/- 1.25%. The product of the conversion separately measured of DHT to 3 alpha diol and 3 alpha diol to 3 alpha diolG was 1.5%, which is not very different than the overall conversion rate of DHT to 3 alpha diolG of 1.4%. This study indicates that the predominant path in this tissue is DHT in equilibrium 3 alpha diol----3 alpha diolG, rather than formation of DHT17 beta G and then 3 alpha reduction to 3 alpha diolG.     

Modulation of porcine thecal cell aromatase activity by human chorionic gonadotropin, progesterone, estradiol-17 beta, and dihydrotestosterone

Porcine thecal cells synthesize estradiol, which may function as an intraovarian regulator of follicular growth. Production of estradiol by granulosa-cell aromatase is modulated by gonadotropins and local steroidal and nonsteroidal factors. Therefore, the effect of human chorionic gonadotropin (hCG) and physiological concentrations of steroids on aromatase activity of the thecal cells was determined. Theca was excised from large porcine follicles (greater than 10 mm diameter) and plated as monolayer cultures in 1 ml of serum-free medium. Twenty-four hours after culture, cells were treated as follows: 1) control; 2) hCG (5 IU); 3) progesterone (P, 3 micrograms), estradiol-17 beta (E, 4 micrograms), or dihydrotestosterone (DHT, 1 microgram); 4) hCG + P, E, or DHT. After 27, 30, 36, 48, and 72 h of culture, media were assessed for levels of P and E. Aromatase activity was determined by a radiometric assay. Levels of P in control media increased from 27 to 72 h. hCH significantly (p less than 0.01) increased P levels from 27 to 72 h of culture. Estrogen decreased (p less than 0.05) P levels at 36, 48, and 72 h compared to controls and also prevented the hCG-induced increase in P levels at these times. DHT significantly increased (p less than 0.05) P levels at 48 and 72 h. DHT + hCG reduced the hCG-associated increase in P concentration at 36 h and 72 h, but enhanced the hCG-induced increase in P levels at 48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thecal cell 3-beta hydroxysteroid dehydrogenase activity: modulation by human chorionic gonadotropin, progesterone, estradiol-17 beta and dihydrotestosterone

Thecal cell steroidogenesis plays a major role in folliculogenesis within the porcine ovary. Accordingly, the effects of physiological concentrations of steroids on 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) were determined. Theca was excised from large porcine follicles and prepared in a monolayer culture in 1 ml of serum-free media. Cells were treated 24 h after culture as follows: (1) control, (2) hCG (5 IU); (3) progesterone (P, 3 micrograms); estradiol-17 beta (E, 4 micrograms); 5 beta-dihydrotestosterone (DHT, 1 microgram); (4) hCG + P or E or DHT. At 3, 6, 12, 24 and 48 h after treatment, media were assessed for P levels. For 3 beta-HSD activity, P formation by microsomal fractions incubated with 1 microM pregnenolone + 5 microM NAD+ for 1 h (37 degrees C) was monitored. Thecal cell P secretion increased from 27 to 72 h. hCG significantly (P less than 0.05) increased P levels after 36 h compared to controls. E or E + hCG decreased P levels at 36, 48, and 72 h and DHT prevented the hCG-induced increase in P secretion. 3 beta-HSD activity in thecal microsomes increased significantly from 27 to 72 h. hCG had little effect on 3 beta-HSD activity compared with controls from 27 to 36 h, but significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. However, P or P + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at all times. In addition, E or E + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. DHT prevented the hCG-induced decrease in 3 beta-HSD activity. In conclusion, porcine thecal secretion of P and microsomal 3 beta-HSD activity increased during 72 h of culture. Paradoxically, the addition of hCG to cultures enhanced media P concentrations but inhibited 3 beta-HSD activity. Further, the addition of E to cultures decreased media concentrations of P while P or E decreased 3 beta-HSD activity. Therefore, paracrine/autocrine effects of locally produced steroids may play a role in modulating thecal cell steroidogenesis.     

The elimination of ryegrass mosaic virus from Lolium multiflorum by meristem tip culture

Meristem tips were cultured from Lolium multiflorum plants infected with ryegrass mosaic virus (RMV). Meristem tips within the size range O'2-fi mm long were cultured on media with or without 2 ,4-D at 1 mg/1. The plants that regenerated in culture showed no symptoms over a 10 month period and no RMV particles were observed by electron microscopy. It was concluded that RMV had been eliminated. The method should prove useful in eliminating the virus from desirable genotypes used as parent material for seed production.     

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and -negative breast cancer cells

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-α (ER-α)-positive MCF-7 cells and ER-α-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-α transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3α, 17β-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin αvβ3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin αvβ3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-α-positive and ER-α-negative breast cancer cells, but by discrete mechanisms.     

Improved Cultivation Systems for Isolation of the Colorado Potato Beetle Spiroplasma

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.