Specific heavy metal labeling of the 3-'terminus of phosphorothioate modified yeast tRNAPhe.

Yeast tRNAPhe containing a phosphorothioate modified -CS-CS-A terminus binds two moles of chloroterpyridineplatinum(II). This result was determined by titrating the tRNA with [3H](terpy)PtCl] Cl, removing excess platinum by cation exchange chromatography, and determining the amount of bound platinum by radiocounting techniques. It has thus been established that adjacent phosphorothioate modified nucleotides can be labeled with an electron dense stain, a necessary requirement for electronmicroscopic sequencing of polynucleotides to become practical.     

Spectroscopic and electrochemical properties of chloro-N-methylprotoporphyrin IX dimethyl ester iron(II), a species related to the intermediate in the formation of N-alkylprotoporphyrins from the cytochrome P-450 prosthetic group

N-alkylporphyrins are formed when certain agents such as 3,5-diethoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine or ethylene interact with cytochrome P-450 in rats. It is likely that the iron protoporphyrin complex in cytochrome P-450 is first alkylated and then demetallated to form the free base N-alkylprotoporphyrins that are observed. An iron complex of N-methylprotoporphyrin IX dimethyl ester, chloro-N-methylprotoporphyrin IX dimethyl ester iron(II), shows the following properties: a double Soret band (λ_max = 435 nm, with a shoulder at 390 nm) relatively facile reduction (E_{$\text{1}\text{2}$} for Fe(III)/Fe(II) of 0.385 V vs Ag/AgCl in acetonitrile) and facile demetallation by acid or good nucleophiles such as thiophenol. A knowledge of such properties should be useful in determining the mechanism of formation of N-alkylprotoporphyrins in vivo.     

Multiple matings in the blue gourami, Trichogaster trichopterus (Pisces,Belontiidae)

The reproductive behaviour of the blue gourami, Trichogaster trichopterus, was examined in 15 bisexual pairs and 22 trios maintained in 75-litre aquaria for 23 days. During this time, 74 spawning episodes occurred among 26 different pairs and trios. Sixty-nine of these spawnings were directly observed. Both males and females were capable of mating repeatedly with the same or different partners with as little as 20 to 24 h separating successive spawnings. Females often mated with two males in the same afternoon, while only one male succeeded in mating with two females in one day. It is concluded that blue gouramis probably do not form pair bonds and that both sexes will mate with differet partners when the physical environment permits two or more males to maintain territories in the same aquarium.     

Modulation of prostaglandin of the renal vascular action of arginne vasopressin

To determine whether the renal vascular effect of arginine vasopressin (AVP) is modulated by renal prostaglandin E₂ (PGE₂) were determined during the infusion of AVP in dogs during control conditions and after the administration of the inhibitor of prostaglandin synthesis, indomethacin. During control conditions, intrarenal administration for 10 min of a dose of AVP calculated to increase arterial renal plasma AVP concentration by 75 pg/ml produced a slight renal vasodilation (p<0.01) and an increase in renal venous plasma concentration of PGE₂. Renal venous PGE₂ concentration during control and AVP infusion averaged 33 ± 7 and 52 ± 12 pg/ml (p<0.05), respectively. After administration of indomethacin, the same dose of AVP induced renal vasoconstriction (p<0.05) and failed to enhance renal venous PGE₂ concentration (9 ± 1 to 8 ± 1 pg/ml). Intrarenal administration of 20 ng/kg. min of AVP for 10 min induced a marked renal vasoconstriction (p<0.01) and increased renal venous plasma PGE₂. Renal venous PGE₂ during control and AVP infusion averaged 31 ± 10 and 121 ± 31 pg/ml (p<0.01), respectively. Administration of the same dose of AVP following indomethacin produced a significantly greater and longer lasting renal vasoconstriction (p<0.01) and failed to increase renal venous plasma PGE₂ (10 ± 1 to 9 ± 1 pg/ml). These results indicate that a concentration of AVP comparable to that observed in several pathophysiological conditions induces a slight renal vasodilation which is mediated by renal prostaglandins. The results also indicate that higher doses of AVP induce renal vasoconstriction and that prostaglandin synthesis induced by AVP attenautes the renal vasoconstriction produced by this peptide.     

Rat lymphocyte subsets: Cellular requirements for the generation of T-cell growth factor

In this study, the cells producing T-cell growth factor (TCGF) in the rat MLR were characterised with respect to the antigens defined by $\text{W}\text{3}\text{13}\text{,}\text{W}\text{3}\text{25}$, and OX8 monoclonal antibodies. Unfractionated lymphocytes and cells depleted of OX8 positive cells were found to be fully capable of producing TCGF, whereas lymphocytes depleted of $\text{W}\text{3}\text{13}\text{and}\text{W}\text{3}\text{25}$ positive cells had lost this ability. Parallel experiments demonstrated that cells selected by the fluorescence-activated cell sorter for the expression of $\text{W}\text{3}\text{13}\text{and}\text{W}\text{3}\text{25}$ defined antigens were potent producers of TCGF. Further studies suggested a functional role for the antigen defined by $\text{W}\text{3}\text{25}$ antibody because the addition of this antibody to a MLR abrogates TCGF production. These findings suggest that the important immunomodulatory functions of $\text{W}\text{3}\text{25}$ positive lymphocytes could be exercised via the synthesis of essential lymphokines.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Kinetic and magnetic resonance studies of substrate binding to galactose oxidase copper(II)

Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate beta-O-methyl-D-galactopyranoside (beta-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN- or F- are used as inhibitors of GOase with beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O2, CN- and beta-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN- binds to the GOase Cu(II) at an equatorial coordination site, as does the F- detected in esr experiments. Thus, the results from the various competition experiments supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.     

Chemotherapy of rodent malaria: transfer of resistance vs mutation

Pyrimethamine-resistant strains of Plasmodium berghei and P. vinckei were produced by exposing populations of erythrocytic parasites to the selection pressure of increasing doses of drug as well as by single-step mutations. Pyrimethamine-sensitive parasites of both rodent plasmodia were found to mutate at a rate of 1–2 × 10^?11 when exposed to a single course of drug therapy, consisting of 15 mg/kg/day for 4 consecutive days, given subcutaneously. Resistance obtained by either method, was found to be stabile for at least 40 passages in the absence of drug pressure, the longest number of passages tested. Parasites exposed to 15 mg/ kg/day were also found to be resistant to 160 mg/kg/day, the maximum dose of pyrimethamine tolerated by the rodent host.Plasmodium berghei chloroquine-sensitive parasites were found to have a mutation rate of 1.5 × 10^?10, when exposed to a single course of chloroquine therapy, consisting of 30 mg/kg/day chloroquine base given for 4 consecutive days, subcutaneously. These parasites were also found to be resistant to 60 mg/kg/day the highest dose of chloroquine tolerated by the rodent host. Chloroquine-resistant strains of P. vinckei could not be developed by a single-step mutation nor by selection by slow increases in drug pressure.Pyrimethamine-resistant strains of P. berghei, whether, the resistance was developed by single-step mutation, or by slowly increasing the pyrimethamine doses over extended periods of time, demonstrated dihydrofolate reductases which were similar in activity, Michaelis constants, and inability to be stimulated by increased concentrations of KCl. The same was found to be true for the dihydrofolate reductases (EC 1.5.1.3) isolated from pyrimethamine-resistant P. vinckei strains. The enzymes isolated from the resistant strains differed in all respects from their sensitive counterparts.Attempts at drug resistance-transfer, using both a biological filter system, and a dual drug resistant system, were both unsuccessful. The origin of all drug resistant strains studied and reported in this paper, can best be explained by the occurrence of mutation, most probably involving the change of a single nucleotide base in the DNA.     

Effects of diverse androgens on the sexual behavior and morphology of castrated male quail

Castrated male Japanese quail were injected for 15 days with 1 mg/day of testosterone propionate (TP), testosterone (T), androstenedione (AE), androsterone (AO), 5α-dihydrotestosterone benzoate (5α-DHTB), or 5β-dihydrotestosterone (5β-DHT), or with oil. Copulation was activated to a significant extent only by TP and T. Strutting was activated only by TP, T, and AE. Proctodeal (foam) glands were well-developed in birds injected with TP, T, AE, or 5α-DHTB. Additional data were obtained following implantation of pellets of crystalline T, AE, AO, or 5α-DHT. T pellets activated copulation, but AO and 5α-DHT pellets did not. Effects of AE require further study. These results suggest that conversion of androgen to estrogen is necessary for the activation of copulation in the male quail.     

Dual Inhibition of PI3K/AKT and MEK/ERK Pathways Induces Synergistic Antitumor Effects in Diffuse Intrinsic Pontine Glioma Cells

Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with an extremely poor prognosis. Recent studies have shown that platelet-derived growth factor receptor (PDGFR) and its downstream effector pathway, PI3K/AKT/mTOR, are frequently amplified in DIPG, and potential therapies targeting this pathway have emerged. However, the addition of targeted single agents has not been found to improve clinical outcomes in DIPG, and targeting this pathway alone has produced insufficient clinical responses in multiple malignancies investigated, including lung, endometrial, and bladder cancers. Acquired resistance also seems inevitable. Activation of the Ras/Raf/MEK/ERK pathway, which shares many nodes of cross talk with the PI3K/AKT pathway, has been implicated in the development of resistance. In the present study, perifosine, a PI3K/AKT pathway inhibitor, and trametinib, a MEK inhibitor, were combined, and their therapeutic efficacy on DIPG cells was assessed. Growth delay assays were performed with each drug individually or in combination. Here, we show that dual inhibition of PI3K/AKT and MEK/ERK pathways synergistically reduced cell viability. We also reveal that trametinib induced AKT phosphorylation in DIPG cells that could not be effectively attenuated by the addition of perifosine, likely due to the activation of other compensatory mechanisms. The synergistic reduction in cell viability was through the pronounced induction of apoptosis, with some effect from cell cycle arrest. We conclude that the concurrent inhibition of the PI3K/AKT and MEK/ERK pathways may be a potential therapeutic strategy for DIPG.     

The effects of the antiestrogen CI-628 on sexual behavior activated by androgen or estrogen in quail

This series of experiments sought to determine whether conversion of androgen to estrogen is important in the activation of male sexual behavior in quail by seeing if an antiestrogen will block androgen stimulated copulation in this species. Experiment I compared the ability of two antiestrogens, MER-25 (5 mg/day) and CI-628 (2 mg/day), to block estrogen stimulated characteristics in female quail. Both treatments greatly reduced oviduct growth in “photically castrated” females given estradiol benzoate (EB, 50 μg/day), but only CI-628 reduced receptivity in these birds. In Experiment II surgically castrated males given 50 μg/day EB together with 2 mg/day CI-628 were much less receptive than castrated males given EB alone, and in addition copulated in fewer tests. In Experiments III, IV, and V, castrated males given testosterone propionate (TP) together with CI-628 were compared with males given TP alone. The ability of CI-628 to suppress TP-stimulated copulation increased with increasing CI/TP dosage ratio, and at the highest ratio (4:1), CI-628 effectively blocked copulation in five out of seven birds. Those birds that did copulate did so in fewer tests and performed fewer cloacal contact movements. CI-628 had no antiandrogenic effects in these experiments. These results suggest that estrogens may be important active metabolites of testosterone with respect to quail copulation.     

Progesterone antagonism of androgen-dependent marking in gerbils

The role of progesterone (P) as an androgen antagonist in Mongolian gerbil territorial marking was assessed in the present investigation. Twenty-seven mature males were observed for two consecutive weeks in a marking apparatus. On the basis of the average of the two sessions, subjects were matched and assigned to one of three groups. All animals were orchidectomized and tested three weeks after surgery. Three days after a single postoperative session a schedule of hormonal replacement was initiated. Group 1 was administered 640 μg testosterone propionate (TP). Group 2 received 640 μg TP followed immediately by 3 mg P. A third group received 640 μg TP followed by a 1 mg injection of P. Subjects were tested for marking 24 hr after the injection for a cumulative period of 6 wk.Results indicated that (a) castration drastically reduced marking; (b) TP alone and TP + 1 mg P restored the behavior; (c) TP + 3 mg P inhibited its restoration; and (d) a significant interaction was present between hormonal therapy and the 6-wk testing interval. However, all three hormonal treatments restored sebaceous gland dimensions. Results are discussed in terms of a model of hormone-gene action.     

Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.     

Sexual preferences of male hamsters (Mesocricetus auratus) for conspecifics in different endocrine conditions

The behavioral responses of sexually experienced male hamsters toward a pair of anesthetized conspecifics were investigated. Males spent significantly more time licking, sniffing, and mounting neonatally and adult castrated males than intact males. Adult castrated males receiving oil injections were preferred over castrates receiving exogenous testosterone propionate (TP). Ovariectomized females were preferred over intact males, adult castrated males, or spayed females receiving exogenous TP. It was concluded that the absence of an androgen-dependent factor(s) renders an animal more sexually attractive.     

Effect of vitamin D and its metabolites on developing myogenic cultures

Growth, protein synthesis and expression of creatine kinase (CK) by embryonic chick myogenic cells are inhibited by vitamin D and certain of its metabolites. 25-OH cholecalciferol was most active in concentrations of 10⁻⁵–10⁻⁶ M, with cholecalciferol and ergocalciferol less active in that order. Ergosterol had no activity of this sort. Inhibition of CK was most marked on the 4th and 5th day of culture and was due to suppression of the appearance of CK-MM and MB. CK-BB was not affected and CK-MB was more affected than CK-BB. Skin fibroblasts by comparison were slightly stimulated in growth at 10⁻⁶ M and much less affected at 10⁻⁵ M than the myogenic cells. It is suggested that vitamin D has a direct effect upon the muscle cell, to cause a selective diminution in the production of certain polypeptides.     

Protamine-DNA association in mammalian spermatozoa

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.     

Crossed immunoelectrophoresis from sodium dodecyl sulfate-polyacrylamide gels into antibody-containing agarose: an improved method for evaluation of the number of immunochemical determinants in polypeptides, with reference to spectrin.

A simple method is described for performing crossed immunoelectrophoresis into antibody-containing agarose when the first-dimension gel contains peptides separated by electrophoresis in sodium dodecyl sulfate. Artifacts produced by sodium dodecyl sulfate are avoided by incorporation of Triton X-100 in the agarose layer. Peptides are located by prestaining (before SDS-acrylamide electrophoresis) with the cycloheptylamylose complex of fluorescamine. Injection of ink into prestained peptide bands produces a line extending from the peptide band location to its precipitin arc, thereby allowing unambiguous assignment of arcs to peptides in situations where peptide bands are not widely separated. The utility of this procedure is illustrated for the erythrocyte membrane protein spectrin.