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Summary Incubation of rat liver chromatin with NAD + resulted in an inhibition of the Ca 2+ , Mg 2+ -dependent endonuclease while the Mg 2+ -dependent endonuclease was not affected. To establish that the endonuclease was blocked directly by adenosine diphosphate ribosylation purified enzymes were used in the reaction mixture. The following ingredients were required in order to demonstrate the inhibitory effect: partially purified Ca 2+ , Mg 2+ -dependent endonuclease, purified poly(adenosine diphosphate ribose) synthetase, NAD + and DNA.

Poly(adenosine diphosphate ribulose) [poly(ADP-Rib)] glycohydrolase activity was determined by measuring the amount of ADP-Rib hydrolyzed from polymers of ADP-Rib as substrate. In principle, the method consists of incubating oligomers or polymers of [14C]ADP-Rib with testis glycohydrolase. The reaction was stopped by the addition of a suspension of Dowex 1X-2 formate in H2O (1:3, vv) which adsorbed monomers and oligomers of ADP-Rib. The adsorbed [14C]ADP-Rib was selectively extracted from the resin with 6 m formic acid. The amount of [14C]ADP-Rib was estimated by measuring the radioactivity in aliquots of formic acid extract. Oligomers or polymers of ADP-Rib can be utilized as substrates since the reaction rates were the same with either compound. A method to determine phosphodiesterase and glycohydrolase activities was established. These two enzymic activities were distinguished by treating the products of the reactions with alkaline phosphatase and by differential extraction of the adsorbed reaction products on Dowex with 0.5 m and 6 m formic acid.

Inkubation von isolierten Rattenhodenzellkernen mit Nikotinamidadenindinukleotid induzierte die Bildung von Polyadenosindiphosphatribose, eine Hemmung der Ca2+, Mg2+-abhangigen, alkalischen Endonuklease und paradoxerweise eine Stimulation der Mg2+-abhangigen, sauren Endonuklease.

Oocytes isolated from mature follicles undergo spontaneous maturation when cultured in vitro. Forskolin, an adenylate cyclase stimulator, inhibited resumption of meiosis of cumulus-free mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 85% of the oocytes treated by forskolin at concentrations of 20 micrograms/ml and higher. The inhibiting effect of forskolin was dose-dependent and reversible. FSH, LH, FSH plus LH, estrogen, progesterone, and estrogen plus progesterone did not reverse the block induced by forskolin in cumulus-free and cumulus-enclosed oocytes. The present results suggest that intracellular cAMP may play a role in the regulation of oocyte maturation.

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 {mu}M. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with ({gamma}-{sup 32}P)ATP and ({gamma}-{sup 32}P)GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with ({gamma}-{sup 32}P)ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With ({gamma}-{sup 32}P)GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.

Sera and cervical fluids of some infertile subjects of undetermined etiology contain antisperm antibodies inducing sperm agglutination and/or immobilization. These antibodies might be the underlying basis for the infertile condition (1–5). Non-aggl utinating or nonimmobilizing antisperm antibodies might also be involved in some cases of infertility by affecting in some unknown manner the fertilizing capacity of sperm or interfere with sperm-egg interaction. To identify the components constituting human sperm membrane that may act as antigens, we have raised monoclonal antibodies against human sperm membrane proteins. One of the cell lines produced antibodies that agglutinate human sperm (6). The interacting protein had an estimated mol. wt. of 84 kDa. In the present study, we have cultivated a second mouse fused spleen cell line raised against human sperm membrane proteins, producing antibodies that induce sperm agglutination. Localization of the antigen on the sperm surface and on colon carcinomatous cells was determined by an immunocytochemical method. Studies on the interaction of the antibodies with purified samples of carcinoembryonic antigen were carried out.

The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.

The presence of choriogonadotropin- and alpha-subunit-like materials in two species of bacteria identified as Staphylococcus simulans and Streptococcus faecalis have been demonstrated by the indirect fluorescein-labeled and the indirect peroxidase-labeled immunocytochemical techniques, utilizing antiserum for human choriogonadotropin, for its alpha and beta-subunits and the bera-subunit COOH-terminal peptide. The bacteria were originally isolated from the urine of two patients with advanced forms of cancer. Chromatography done on the water-soluble extract of acetone powder preparations of the bacterial cultures revealed the presence of a material similar to the complete trophoblastic hormone and to its beta-subunit in the culture media of S. simulans, and to the beta-subunit in the media of S. faecalis. No free alpha-subunit was detectable. Furthermore, the choriogonadotropin-like factor demonstrated biological activity in in vivo assay systems. From the present results, it can be concluded that some species of "cancer-associated" bacteria can synthesize a human trophoblastic hormone-like glycoprotein with physicochemical properties similar to those of the human trophoblastic hormone that is biologically active and that is either released complete or as one of its subunits in the culture media.

In the present communication, data will be presented to show that a strain of Streptococcus faecalis produces a factor having immunoreactivity to antiserum raised against the β-subunit of human chorionic gonadotropin (hCG). The partially purified factor displaces the β-subunit in the homologous CGs radioimmunoassay (RIA) system. In addition, the expression of CG/CGs, as well as of CGα immunoreactive substances in the microorganism, was demonstrated by immunohistochemical techniques.

Publisher Summary This chapter discusses biochemical transmitters regulating the arrest and resumption of meiosis in oocytes. Germ cells migrate to the genital ridge from the yolk sac region during early embryonic development. In the genital ridge, the female germ cells start to divide and differentiate into oogonia. They enter meiosis and become primary oocytes. Nuclear division progresses to the diplotene stage of the first meiotic prophase and is arrested. The chromosomes decondense and are distributed diffusely throughout the oocyte nucleus. Progression of meiosis to the diplotene stage occurs before or shortly after birth. The oocytes may remain arrested at the dictyate stage for a prolonged period. Subsequently, a follicle develops enclosing the oocyte that contains a large clear nucleus designated as the germinal vesicle. The resumption of meiosis follows a sequence of programmed events while the oocytes are situated within the preovulatory follicles. The process is designated as oocyte maturation and is characterized by a series of biochemical, morphological, and functional changes that take place within the nucleus, highlighted by various events: (1) dissolution of the nuclear membranes manifested as germinal vesicle breakdown (GVBD), (2) chromatin condensation and the formation of distinct chromosomes, (3) formation of the first meiotic spindle, (4) translocation of the spindle to the peripheral region, (5) formation and extrusion of the first polar body, (6) formation and positioning of the second meiotic division, (7) rearrest at the second metaphase.

Production of a factor resembling chorionic gonadotropin (CG) by a microorganism named Progenitor cryptocides was reported by Livingston and Livingston (1974) and Cohen and Strampp (1976). The factor was tentatively identified as a CG-like substance based on hemagglutination inhibition assay, radioimmunoassay (RIA), and radio-receptor assay (RRA) methods. Recently, with use of immunocytochemical techniques, Acevedo and associates (1978, 1979) and Slifkin et al. (1979) demonstrated that CG-like immunoreactive substance is present in the cell wall of several microorganisms which were isolated from patients bearing malignant neoplasms. Furthermore, Richert and Ryan (1977a) reported that the media in which Pseudomonas maltophilia were cultured may contain a protein which cross-reacts with antisera to hCG and hCGB. Other microorganisms which were reported to produce or contain CG-like factor are Escherichia Coli (Cohen and Strampp, 1976), Staphylococcus epidermidis (Affronti et al., 1977a, b; Affronti and Charoenvit, 1979) and Staphylococcus faecalis (Koide et al., 1979).

The characteristics of the reaction of 11β,17α-dihydroxy-2,4-pregnadiene 3,20-dione-21-al (21-dehydroprednisolone) with amino acids under varying conditions were investigated. The rate of the reaction of 21-dehydroprednisolone with alanine was accelerated with increasing concentration of reactants, at alkaline pH, with elevation of temperature and in the presence of phosphates. The products of the reactions were steroid-amino acid complexes (Schiff bases), 21-amino or 21-imino corticosteroid derivatives and keto acids. The 21-amino- or 21-imino-prednisolone may react with excess 21-dehydroprednisolone to form a diprednisolone amine.

The effects of intrauterine application of actinomycin D and calf thymus histones on the akaline phosphatase activity and histological changes of the uteri of castrated rats stimulated with 17β-estradiol were studied. The uterine phosphatase activity induced with 17β-estradiol was suppressed nearly to the level of castrated rats at high doses of actinomycin D (6 μg/horn). Although the epithelial cells were completely denuded, histochemical localization of phosphatase activity was demonstrated in the blood vessels, glandular epithelium and stroma. Lower doses of actinomycin D inhibited the height of the epithelial cells induced by 17β- estradiol and an unexplained rise in the phosphatase activity above the control level was observed. Calf thymus histones administered into the uterine cavity incited an acute inflammatory reaction and did not inhibit the increase in uterine alkaline phosphatase induced by 17β- estradiol. (Endocrinology 82: 1069, 1968)

The influence of intrauterine instillation of uterine RNA on protein, lipid and RNA synthesis in uteri of ovariectomized rats was studied. Extensive purification of the uterine RNA was performed to exclude any residual extradiol-17β in the preparations. RNA was isolated from the uteri of estrogen-stimulated rats by phenol fractionation or diethyloxydifonnate treatment and washed twice with 95 per cent ethanol. The RNA preparations (8–10mg) were dissolved in 20 ml of saline and tested for estrogen-like activity by a single instillation into ligated uterine horns of ovariectomized rats. [3H]uridine (100μCi) and [14C]glycine (25 μCi) were administered intravenously to ovariectomized rats at various times after the intrauterine instillation of RNA. Rats were killed 30 min after the administration of uridine and 30 or 60 min after glycine. The incorporation of radioactivity into total RNA, lipids and proteins was determined. Manipulative procedures such as ligation of uterine horns and/or intrauterine instillation of saline stimulated uterine RNA synthesis. When uterine and liver RNA obtained from estrogenstimulated rats and washed with ethanol but not with ether were administered, however, the increase in RNA synthesis was significantly greater than the procedural control values at 4 and 8 h after the intrauterine instillation. To exclude any residual estrogen the uterine RNA preparation was extracted with ether 6 times. When the sediment from the organic phase was dissolved in oil and assayed, it stimulated uterine RNA synthesis. The uterine RNA obtained from control non-stimulated ovariectomized rats stimulated RNA synthesis. However, the values obtained were equivalent to the ones observed with saline instillation or following ligation of the uterine horns. Uterine lipid synthesis was increased, also, by ligation or intrauterine instillation of saline. The increase in lipid synthesis initiated by uterine RNA was equivalent to the procedural control values. Protein synthesis, however, was increased by uterine RNA obtained after ethanol washing and the sediment of the ether phase. To analyze for the possible presence of residual estradiol-17β in the uterine RNA preparations, the contents of estradiol-17β in the preparations were determined.[3H] or [14C]estradiol-17β (10 μg) was administered intravenously to ovariectomized rats. The RNA were prepared from the uterus, kidney and liver 4 h after the hormone treatment. The content of estradiol-17β in the RNA preparations as calculated from the radioactivity ranged from 0.05 to 3.5 pg per 10 μg of RNA. It was determined that a single dose of 1 pg of estradiol-17β administered by intrauterine instillation stimulated RNA synthesis at 4h, increased slightly lipid synthesis at 30 min and accelerated protein synthesis at 4 h. The present results reveal that uterine and liver RNA extracted with phenol and washed with ethanol but not with ether contains small amounts of estrogen which may account for the stimulatory action of such extracts. After repeated washings of RNA with ether the estrogen content was reduced to less than 10−2pg of estradiol-17β per 10μg RNA. The ether-extracted uterine or liver RNA did not stimulate RNA synthesis, but did increase protein synthesis at 24 and 48 h after instillation. RNA extracted from estrogen-free proliferative uteri induced by intra-uterine pressure possesses similar biologic activity. These results indicate that the ability to stimulate protein synthesis may be an intrinsic property of RNA from proliferating uteri.

Labeled steroids were incubated with erythrocytes of human subjects, dogs, and Pekin ducks, suspended in saline and extracted with organic solvents. The radio-activity in the saline washings, organic phase and aqueous (residual) fractions was determined. The amounts of various steroids in the fractions obtained from human and dog erythrocytes were similar. A distinct difference in steroid content was observed between the duck and the mammalian erythrocytes. The organic phase and aqueous fraction obtained from the human and dog erythrocytes had a higher content of aldosterone, progesterone, testosterone, dehydroepiandrosterone and 21-dehydrocortisol than those obtained from duck erythrocytes. On the other hand, both fractions from duck erythrocytes contained larger quantities of estrogens and glucocorticoids. When 21-dehydrocortisol was incubated with various erythrocyte suspensions, significant conversion to cortisol occurred only with duck erythrocytes.

DNA obtained after complete removal of the proteins from NAD-treated rat liver chromatin possessed template capacity equivalent to that obtained from untreated chromatin. The stepwise extraction of proteins from chromatin affected a gradual removal of labeled ADPR and a parallel decrease of the inhibition. The results of the present study suggest that the inhibition of the template capacity for DNA synthesis following preincubation of chromatin with NAD is dependent upon ADP-ribosylation of the associated proteins. The template capacity of chromatin and nucleoprotein complex (Weiss preparation) for DNA synthesis was inhibited, whereas the capacity for RNA synthesis was apparently not affected.

Es wurde Actinomycin D s.c. an junge Ratten verabreicht. Beobachtet wurde die Wirkung der Substanz auf Korpergewicht, Uterusgewicht sowie auf die alkalische Phosphataseaktivitat des Uterus vor und nach 17β-Oestradiol Stimulation. Actinomycin D (10µg/40 g/Ratte) verhinderte sowohl bei mit Oestradiol behandelten Tieren, wie auch bei den Kontrollen ein Ansteigen des Korper- oder Uterusgewichts. Der Einfluss auf die durch Oestradiol induzierte alkalische Phosphataseaktivitat des Uterus blieb minimal und bedeutungslos.

[M- 14 C]-cortisol was administered intraperitoneally to adrenalectomized female rats. After 15 min the rats were killed by decapitation. Liver homogenates prepared in 70% methanol were centrifuged. The supernatant was extracted with organic solvents. The radioactivity remaining in the aqueous fraction was about 14% of the total counts in the original supernatant. The metabolites in the aqueous fraction were identified as 3α,11β,17α-trihydroxy-5α-pregnan-20-one-21-yl-sulphate] and 3β,11β,17α-trihydroxy-5α-pregnan-20-one-21-yl-sulphate. The ratio of the former to the latter was 7:3. Two of the U.V. absorbing materials which were eluted in the steroid fraction after gel filtration through Sephadex column were identified as inosine and hypoxanthine.

The reaction of 21-dehydrocorticosteroids with amino acids or peptides in phosphate buffer was studied by measuring the fluorescence spectra of the mixture and by identifying the products formed. Changes in the fluorescence spectra suggest that the steroid reacted with the α-amino group with the formation of a labile Schiff base intermediate. The products of the reaction of 21-dehydrocortisol and glycylglycine were identified as cortisol-21-amine and N -glyoxylglycine.

The enzymic hydrolysis of various steroid phosphates incubated in plasma from normal and pregnant women was determined. Cortisol 21-phosphate was hydrolyzed by plasma alkaline phosphatase and testosterone 17β-phosphate by plasma acid phosphatase. The other steroid phosphates tested were not hydrolyzed. (Endocrinology 82: 1062, 1968)

A study was conducted to observe the effect of cortisol on the melting profile (thermal denaturation) of DNA isolated from human term placenta. The melting temperatures of the DNA in 0.7 mM phosphate buffer, pH 6.9, and in 0.15M NaCl, 0.015M trisodium citrate solution, pH 7.2, were61.5 and 86.5 C, respectively. Cortisol at concentrations of 1.4–140 μM did not affect the melting profile of the placental or calf thymus DNA. It remained unaltered when the experiments were conducted in different buffers with varying ionic strengths. (Endocrinology 84: 683, 1969)

SummaryThe influence of thyroxine on the incorporation of uridine-3H into uterine acid soluble and RNA fractions and on nuclear RNA polymerase activity of ovariectomized-thyroidectomized rats was studied. The incorporation of uridine-3H into uterine RNA of untreated ovariectomized-thyroidectomized rats was higher than that of untreated ovariectomized rats which was further elevated by thyroxine and 17β-estradiol administration. A potentiating effect was observed following the administration of both hormones which was inhibited by actinomycin D.The uterine nuclear RNA polymerase activity of untreated ovariectomized-thy-roidectomized rats was higher than that of ovariectomized rats. Thyroxine administered to ovariectomized-thyroidectomized rats depressed the polymerase activity and 17β-estradiol administration of these rats did not influence the polymerase activity. The results of the present study suggest that thyroxine influences uterine RNA synthesis and potentiates the stimulatory effect of 17β-estradiol.

Lopez-Redondo et al. use cryo-EM and molecular dynamics to evaluate the effects of Zn2+ binding on the conformation and dynamics of the zinc/proton antiporter YiiP. Removal of Zn2+ leads to enhanced conformational dynamics and causes a transition to an intermediate state in the transport cycle., YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Simultaneous addition of an aliquot of body fluid obtained from the surf clam, Spisula solidissima, enhanced oocyte germinal vesicle breakdown induced with serotonin but not with KCl. When the body fluid and serotonin were added sequentially to the oocytes, potentiation did not occur. Body fluids of both males and females were effective at a 200-fold dilution. The factor is stable when treated with heat, acid, base, trypsin and pronase. It is hydrophobic and not dialyzable through tubing with a molecular weight cutoff of 1000 daltons. The factor is probably not a protein.

Bacteria capable of synthesizing and incorporating in their cell membranes a material with immunological, physicochemical, and in vitro and in vivo biological properties similar to those of the human trophoblastic hormone, chorionic gonadotropin (CG), have been isolated from human tissues and urine. The findings are of great biological importance since to date, each one of the strains of bacteria demonstrating the capability to express the CG-like material was isolated from a patient with clinical cancer, and also because it is the first demonstration of biosynthesis of a biologically active “glycoprotein hormone-like” material by procaryotes.In order to establish if morphologic and/or ultrastructural differences exist between a bacterium that synthesizes CG-like material (CG-producer) and those that do not, transmission electron microscopy studies have been done in eight strains of CG-producers. There were five strains of Staphylococcus epidermidis and single strains of Staphylococcus simulans, Streptococcus faecalis, and a bacterium tentatively identified as aerobic “coryneform-like.”

Gonadotropin, durch Fallung mit Aceton und Ammoniumsulfat aus menschlicher Placenta (HPG) isoliert und durch Chromatographie an CM- und FEA-Cellulose sowie durch Gelfiltration an Sephadex G-100 gereinigt, ergab Fraktionen, die in hoher Dosis bei jungen Mausen Blutungen im Ovar hervorriefen, was zeigt, dass GPH eine FSH-ahnliche Wirkung bewirkt.

Die Umwandlung von Kortisol in Kortison und umgekehrt wird nach Injektion von radioaktiven Steroiden bei einem weiblichen Affen (Macaca mulatta) untersucht. Es konnte festgestellt werden, dass im Blut Kortisol relativ rasch in Kortison umgewandelt wurde, wahrend umgekehrt Kortison wesentlich langsamer umgewandelt wird.

Background Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules. Results Stable mRHBDD1 knockdown GC-1 cells were sensitive to apoptotic stimuli, PS341 and UV irradiation. In vitro, they survived and proliferated normally. However, they lost the ability to survive and differentiate in mouse seminiferous tubules. Conclusion Our findings suggest that mRHBDD1 may be associated with mammalian spermatogenesis.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T-cells. A phage-display library of 11,200 novel proteins is designed using the α-KTx scaffold found in 31 scorpion toxins that bind to potassium channels and mokatoxin-1 (moka1) isolated by sorting on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not impact Kv1.1, Kv1.2 or KCa1.1. Thus, moka1 suppresses CD3/28-induced cytokine secretion by T-cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach revealing a unique interaction surface supported on an α-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins. Success with other toxin scaffolds and sorting with cell-surface targets has extended utility of the approach.