Search

We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.

Synthetic short peptides containing only the nuclear localization signal (NLS) direct the transport of nonnuclear proteins into the nucleus. As a conjugate of the synthetic peptide with immunoglobulin M (IgM) did not enter the nucleus, there was believed to be a size limit for nuclear transport of NLS-conjugated proteins. However, we found that IgM conjugated with purified nucleoplasmin, a nuclear protein of Xenopus oocytes, rapidly accumulated in the nucleus. For direct comparison with the short peptide, we prepared a long peptide containing the NLS and its flanking sequences of SV40 large T-antigen and its mutated long peptide, in which possible phosphorylation sites located at the amino terminal of the NLS were changed to alanine. Kinetic experiments showed that wild-type long peptide-IgM conjugates were almost entirely taken up into the nucleus within 30 min after their injection, whereas almost 60 min was required for the mutated long peptide-IgM conjugates to enter the nucleus of all the cells examined, and there was no apparent accumulation of short peptide-IgM conjugates in the nucleus within 60 min. These results indicate that even when the kinetics of transport are affected by amino acid substitutions, the long peptide directs the transport of large molecules such as IgM into the nucleus.

Excellent results have been obtained for the Fmoc solid-phase syntheses of peptides using the activating reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU). Activation occurs very rapidly in N,N-dimethylformamide and N-methyl-pyrrolidone, optimal solvents for peptide-resin solvation. It has been observed that complete coupling reactions occur in only 10-30 min. Residues such as Arg, Ile, Leu and Val, which often require double coupling by other activation methods, react with high efficiency by single coupling when HBTU is used. The Fmoc/HBTU chemistry has recently been applied to the peptide synthesizers. The incorporation of trityl side-chain protection for Fmoc-Asn and Fmoc-Gln further enhances coupling efficiencies in difficult sequences.

In folklore medicine, extracts of the leaves of the subtropical plant Catharanthus roseus (L.) G. Don (sometimes known as Madagascar periwinkle) were reputed to be useful in the treatment of diabetes. This review describes how attempts to verify the antidiabetic properties of the extracts led instead to the discovery and isolation of two complex indole alkaloids, vinblastine and vincristine, which are used in the clinical treatment of a variety of cancers. The two alkaloids, although structurally almost identical, nevertheless differ markedly in the type of tumors that they affect and in their toxic properties. These and related alkaloids have been the subject of many pharmacological and biochemical investigations both in vivo and in vitro in the search for improved cancer treatments. A model system used in these studies, a transplantable lymphoma in Noble strain rats designated Nb2 node, has serendipitously led to the development of a highly sensitive and specific bioassay for lactogenic hormones.

Two forms of local cutaneous graft-versus-host reactions were used to examine the in vivo activity of cytolytic T cells in a large number of antigen systems and mouse strain combinations. In immune lymphocyte transfer reactions (TrRs), CTL were injected intradermally into allogeneic hosts to which they were sensitized; in bystander reactions (ByRs), CTL were mixed with target cells and the mixture injected into hosts syngeneic to the CTL. Both reactions frequently culminate in full-thickness skin destruction. However, CTL highly active in cell-mediated lympholysis assays in vitro sometimes failed to induce significant reactions in vivo, and CTL with negligible CML activity often induced severe, necrotizing lesions. In addition, Clone 58, a non-MHC-specific CD8+ clone that originated from cells extracted from a sponge matrix allograft, lost its CML activity but continued to induce necrotizing TrRs and ByRs. Insofar as these reactions may exemplify the specific (TrR) and nonspecific (ByR) tissue injury that occurs in the rejection process, these findings question the reliability of CML for predicting the ability of CTL to induce the tissue destruction seen in allograft rejection.

9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.

Children with Down syndrome (DS) suffer an increased incidence of severe viral and bacterial infections particularly during the first 5 years of life. Unfortunately, few studies have been performed on the immune systems of young children with Down syndrome. Peripheral blood mononuclear cells (PBMC) from a group of non-institutionalized children less than 6 years of age were studied and compared with PBMC from age-matched controls. The children with DS had reduced numbers of circulating OKT4+ (helper/inducer) T cells and a significantly depressed ratio of OKT4+ to OKT8+ (suppressor/cytotoxic) T cells. PBMC from the DS subjects exhibited reduced proliferative responses to phytohemagglutinin and to an optimal concentration of concanavalin A (Con A), but normal responses to suboptimal doses of Con A and pokeweek mitogen. PBMC from young children with DS appeared to produce normal levels of interleukin-2 (IL-2). These findings provide evidence that the primary immune defect in DS is in part a depressed number and function of helper T cells. They also indicate that IL-2 production may not be defective in DS, but rather that the mechanism for response to IL-2 may be faulty.

Transplantable tumor lines were previously established from a variety of estrogen-induced tumors in Nb rats, including tumors of the adrenal cervix, salivary gland, and pancreas, a lymphoma, and a liposarcoma. Spontaneous tumors, however, were found to arise in untreated females and showed the same characteristics. Tumor growth was dependent upon or influenced by estrogen when assessed in estrogenized and unconditioned hosts. Intermitten estrogenization was effective, but tumor growth took place more slowly. The type of response observed led to a new classification of five types of hormone-responsive tumors including tumors inhibited by estrogen. Estrogen-dependent tumor cells might remain dormant indefinitely and not grow in unconditioned animals until stimulated to grow by estrogen. The growth rate of hormone-dependent adrenal carcinomas was related to the amount of estrogen. Tumor growth started more rapidly in the presence of low estrogen dose levels in old rats used as hosts than it did in young rats. Breast carcinomas required the largest amount of estrogen for growth, whereas ovarian thecomas would grow in normal females but not in males. The growth rate in conditioned hosts of most transplanted tumors (some have maintained hormone dependency over 10 years) increased over successive generations. Progression, however, towards a more autonomous state after repeated transplantations was remarkably slow, and a sudden change to autonomy was rarely noted. In contrast, transplants of 9,12-dimethylbenz(a)anthracene-induced mammary carcinomas progressed rapidly to autonomy. Fould's concept of progression (2, 3) has been discussed but the described classification of tumors under hormone influence apparently allows a more detailed analysis of definition of different types of progression.

The replication of Nb 2 Node rat lymphoma cells in suspension culture is specifically stimulated by lactogenic hormones. Human (hPRL), ovine, bovine, and rat PRLs stimulated replication in a dose-dependent manner in the concentration range of 10 pg/ml to 1 ng/ml. Human, ovine, and bovine placental lactogens were similarly active. In addition, cell replication was stimulated by human GH (hGH), which is known to have lactogenic activity. Other hormones and growth factors examined were inactive. The growth stimulatory effects of hPRL and hGH were completely inhibited when excess anti-hPRL and anti-hGH, respectively, were added to the medium. A bioassay based on the response of the Nb 2 Node lymphoma cells to lactogenic hormones has been developed. Human serum stimulated cell replication. The effect was completely abolished if excess antibodies to both hPRL and hGH were present. The stimulation obtained with a number of human serum samples correlated very well with the sum of the hPRL and hGH concentrations in the sera, as determined by RIA (r = 0.95; P < 0.001). The concentrations of either hPRL or hGH in human serum could be individually determined by specifically blocking the growth stimulatory effect of the other hormone by adding excess anti-hGH or anti-hPRl. The sensitivity of this bioassay for PRL and hGH in serum exceeds that of RIAs.

Down's syndrome (DS) is associated with respiratory infections, lymphoblastic leukemia and a mortality rate that is elevated particularly during the first 5 years of life. Unfortunately, most studies on immune function in DS have been performed on adults or older children, the results of which may be not relevant to a primary immune defect in DS since maturation of the immune system and compensatory mechanisms may mask to a certain degree primary immune defects which are associated with this syndrome. In the present study, several immune and hematologic parameters were studied in DS children under 6 years of age and in age-matched controls. The children with DS exhibited a reduced number of lymphocytes while the total number of white blood cells was normal. In addition, the children with DS had reduced numbers of circulating OKT4+ cells and a significantly depressed ratio of OKT4+ to OKT8+ cells, but their overall proportion of T-cells was normal. Normal NK activity was observed and cells from DS children were no more sensitive to the augmenting effects of alpha interferon than cells from control children. Hematocrit levels were slightly elevated in the DS group while plasma zinc levels appeared to be normal in this group.

Using a fluorometric assay, nonspecific proteolytic activity and plasminogen activator were measured in transplantable tumors of the dorsal prostate of Nb rats. Nonspecific proteolytic activity in prostatic tumors did not differ significantly from that measured in normal dorsal prostate, whereas plasminogen activator activity, undetectable in the latter tissue, was readily measurable in the tumors. Furthermore, plasminogen activator activity in prostatic tumors characterized by hormone-insensitive growth was 8-fold higher than in tumors characterized by androgen-stimulated growth. In both typess of tumors, the plasminogen activator activity per mg protein was highest in the lysosomal fractions. The results indicate that plasminogen activator may be a useful marker for discriminating between androgen-stimulated and autonomous prostatic tumors.

In vitro and in vivo-generated cytotoxic T lymphocytes (CTL) specific for major and minor histocompatibility antigens evoked antigen-specific full-thickness skin necrosis when injected intradermally into allogeneic mice in a variety of strain combinations. In addition, CTL-target-cell mixtures injected intradermally into hosts syngeneic to the CTL also evoked destruction of host tissue. These "innocent bystander" reactions were evoked with alloreactive CTL as well as with CTL directed against hapten (TNP)-modified and virus (influenza A)-infected target cells. Unlike the direct reactions, the bystander reactions in histocompatibility-antigen systems occurred in spite of H-2 incompatibility of the CTL, admixed target cells, and the hosts. One explanation for these results, currently under investigation, is that some bystander reactions may occur without MHC restriction. In aggregate, our findings indicate that nonspecific as well as antigen-specific reactions initiated by CTL-target-cell interactions may contribute to tissue destruction in allograft rejection, in severe forms of delayed-type hypersensitivity, and in certain viral infections.

Cytotoxic T lymphocytes generated against epidermal alloantigen-1 (Epa-1), a tissue-restricted, non-H-2 alloantigen that is a target-cell determinant of both skin allograft rejection and cutaneous graft-versus-host reactions, directly produce full-thickness ulcerative skin lesions in Epa-1+ mice. Anti-Epa-1 CTL also indirectly cause extensive damage of "innocent bystander" tissue when injected admixed with Epa-1+ target cells into the skin of Epa-1- hosts. Unlike the direct destruction of host tissue by CTL in "immune lymphocyte transfer reactions" (TrR), "bystander reactions" (ByR) apparently are initiated by the release of lymphokines that recruit host inflammatory cells to the injection site. Treatment of CTL with cyclosporine in vitro prevents their production of lymphokines like interleukin 2 but has no effect on cell-mediated cytotoxicity. However, little is known about the function of CsA-treated CTL in vivo. We confirmed the differential effect of CsA on CTL function in vitro with bulk-culture anti-Epa-1 CTL-CsA pretreatment of CTL abrogated IL-2 production but did not affect CMC. Moreover, we found that CsA pretreatment did not affect the ability of CTL to evoke TrR, nor did it significantly impair their ability to mediate ByR. Therefore, when CTL are treated with CsA in such a way that they lose their capacity to produce IL-2, their cytotoxic activity in vitro as well as their ability to directly and indirectly mediate tissue destruction in vivo are left intact. These results suggest that the ability of CTL to mediate allograft rejection is not dependent on their ability to produce IL-2 and that CMC plays a role in the rejection process.

The bladder epithelium of female Nb rats was markedly stimulated by treatment with pellets of testosterone propionate. Estrone treatment simultaneously reduced the incidence of bladder changes to approximately 50 per cent although estrone alone had no effect on bladder weights. The bladders of male rats were less responsive to hormones and were stimulated only by treatment with estrogen + testosterone. Massive papillary hyperplasia of the uroepithelium that extended into the kidney pelvis could be produced by hormones in the absence of calculi. Transplants of hyperplastic bladder epithelium usually resulted in small pseudo-bladders, but in one case a transplantable squamous cell carcinoma developed.

A transplantable prostatic adenocarcinoma derived from the dorsal lobe of the prostate gland of an Nb rat was analyzed for the concentration of nuclear androgen-binding sites and the presence of acid phosphatase activity. When extracts of nuclei from normal prostatic tissue were labelled with [1,2-3H]dihydrotestosterone in the absence and presence of competitor, two types of specific binding were observed: type 1 was characterized by an association constant (Ka) of 6 x 10(7) M-1 and involved a molecule that was excluded from Sephadex G-200; type 2 was characterized by a Ka of 3 x 10(8) M-1 and depended on a binding component that was retained by Sephadex G-200. Nuclei from androgen-stimulated tumors contained reduced concentrations of both androgen-binding components, whereas nuclei from autonomous tumors had only a trace amount of type 1 sites and were entirely devoid of type 2 sites. In all tumors the acid phosphatase activity per mg of protein was markedly elevated. Relative to normal, the activity of this enzyme was 140% and 350% higher in androgen-stimulated and autonomous tumors, respectively. These findings indicate that prostatic tumors are characterized by a decrease in nuclear androgen-binding, and an increase in specific activity of acid phosphatase, and also that such changes are more pronounced in autonomous than in androgen-stimulated tumors.

Carcinoma in the dorsal prostate of Nb rats was produced by prolonged treatment with androgen or androgen plus estrogen. The most effective combination was androgen followed by estrogen, resulting in an incidence of 50% in 10 rats. In such a case, the rats did not show evidence of androgenization. Transplants were usually not sensitive to hormones, but an estrogen-dependent tumor (Nb-2Pr-E) was successfully transplanted in estrogenized hosts. Cells of estrogen-dependent tumors did not grow in males but remained dormant and could be stimulated to grow after many months by estrogen treatment. The resulting tumors remained estrogen dependent, with one exception which was autonomous. Tumors regressed following estrogen removal but eventually showed a spontaneous regrowth which was then found to be autonomous. In the case of growing tumors, the reduction of estrogen to subthreshold doses for maximal growth allowed stationary or only slow growth of the tumors, but these remained estrogen dependent. Estrogen-dependent tumors responded to tamoxifen treatment by a cessation of tumor growth or regression despite continued estrogen treatment. Tumor regrowth eventually took place after prolonged tamoxifen treatment, but the tumors remained estrogen dependent when transplanted.

Hormonal responsiveness should be considered in terms of at least three homeostatic constraint mechanisms. Observations on the induction of specific responses in rat prostate reveal that certain of these are controlled differentially, and also that immature cells can be modified by low levels of androgen. The conditioning effect of low levels of steroid is further demonstrated by the benefits of fractional hormone-replacement therapy in controlling and delaying tumor progression. A revised approach to endocrine therapy of cancer should be seriously considered. This would entail the fractional replacement of hormone after ablative surgery.

This review outlines the establishment, properties, and use of two lines of cultured Nb rat lymphoma cells. The cultured cells have retained important properties of the cancers of origin, such as dependency on prolactin for growth and a high sensitivity to antineoplastic Vinca alkaloids. The cultures have been useful for defining the hormonal dependency of the lymphomas in the animal and for studying the progression of the lymphomas from hormonal dependency to autonomy. A new, specific and highly sensitive in vitro bioassay for lactogenic hormones has been developed from one of the cultures. The use of the lymphoma cell cultures has revealed unsuspected pharmacological differences between the closely related, clinically useful antineoplastic Vinca alkaloids, vinblastine and vincristine. The lymphoma cell cultures are also useful tools for studying biochemical, cell cycle related events which follow the mitogenic stimulation of cells by a polypeptide growth factor.

Vinblastine (VLB) and vincristine (VCR) have been compared with respect to their inhibitory effects on the growth of cultured Nb2 node rat lymphoma cells and their binding and release by these cells. When examined over a wide concentration range, the two alkaloids were found to be almost equally effective in inhibiting culture growth, providing they were continuously present in the incubation medium. A striking difference was found, however, when the cells were incubated for short periods with the alkaloids and then restored to alkaloid-free medium for further incubation. Cells treated with VCR (5 X 10(-9) g/ml) for 3 h and washed free of extracellular alkaloid failed to resume normal proliferation during a subsequent 48 h incubation period in alkaloid-free medium. In contrast, cells treated with VLB at a higher concentration (5 X 10(-8) g/ml) and for a longer time (7 h), rapidly resumed growth in alkaloid-free medium. Binding studies, using (3H)VLB and (3H)VCR, showed that when cells were incubated continuously with the alkaloids at a given concentration, they bound equal amounts of the drugs. When labelled cells were resuspended in alkaloid-free medium, VLB was released very readily by the cells, whereas VCR was tenaciously retained. The findings indicate that the growth-inhibitory effects of VLB and VCR are related to the amount of alkaloid associated with the cells and that the long-lasting effects of VCR, relative to VLB, are very likely due to the retention of VCR by the cells when the extracellular alkaloid levels decline. The study also suggests that, in the treatment of malignancies, VLB could be more effective if its extracellular levels were maintained, e.g. by continuous administration.

The development of adenocarcinoma of the dorsal prostate of Nb rats following prolonged treatment with pellets of testosterone propionate alone or with esterone allowed the establishment of transplantable tumor models. Whereas most transplants were autonomous and not influenced by hormones, one estrogen dependent tumor was studied in detail. This tumor would only grow in estrogenised hosts, cells remaining dormant in normal animals. Removal of estrogen from animals with growing tumors led to tumor regression but with progression and eventual regrowth of tumors which were autonomous. Replacement with lower doses of estrogen reduced the extent of regression and prevented autonomous change. When a regressed estrogen dependent tumor was exposed to prolonged treatment with testosterone propionate pellets, the eventual regrowth was found to be androgen dependent and would not grow in estrogenised animals. Progression could apparently be directed by this procedure from estrogen to androgen dependency.

alpha-Bungarotoxin, a snake neurotoxin, binds irreversibly and specifically to the acetylcholine receptor isolated from the electroplax of Electrophorus electricus and Torpedo species and has been an important tool in the study of the receptor-ligand binding mechanism. Two distinct kinetic processes have been observed in studies with membranes from E. electricus. A minimum mechanism for the toxin reaction involves (i) the reversible binding of two toxin molecules to the receptor prior to the irreversible formation of toxin receptor complexes and (ii) a toxin-induced conformational change of the receptor which leads to an increase in the affinity of the receptor binding sites for toxin [Hess, G. P., Bulger, J. E., Fu, J.-j. L., Hindy, E. F., & Silberstein, R. J. (1975) Biochem. Biophys. Res. Commun. 64, 1018-1027]. Only one process has been detected in Torpedo membranes. Here, we determine whether the receptors in Torpedo californica and E. electricus membranes have different properties or whether the measurements and their interpretation were responsible for the different results. Two methods which are frequently used in binding studies to separate free and bound toxin, a CM-52 cellulose minicolumn assay and DE-81 filter disk assay, have been compared. The results obtained indicate that the interaction of toxin with receptor from T. californica is similar to that observed with receptor from E. electricus. The apparent differences which have been reported in the literature are shown to have arisen from the design of the experiments in which T. californica membranes were used.

PRL and other lactogenic hormones are potent mitogens in a lymphoma cell line derived from a lymph node of an estrogenized Noble (Nb) rat. The present study demonstrates that these cells (designated Nb2 node) possess receptors that bind only lactogenic hormones. There are approximately 12,000 receptor sites per cell. The kinetics of binding of [125I]iodo-PRL exhibited by Nb2 lymphoma cells is unusual in that PRL binding follows a biphasic pattern. Binding of [125I]iodo-PRL reaches a maximum in 1 h at 37 C, followed by a rapid decline. This pattern was not observed if binding was carried out in the presence of chloroquine, a lysosomotropic agent, or if cell homogenate was used for binding. We also examined the relationship between receptor occupancy and the magnitude of PRL response in these cells. Maximal growth stimulation by PRL occurs when only 35% of the maximal binding of PRL is achieved, suggesting that a majority of the PRL binding sites may be spare receptors. This study also revealed that the dissociation constant (Kd) of the PRL receptors in Nb2 cells is 75 pM, approximately 20-fold higher than that of the receptors in other cell types. PRL at 6 pM produces a half-maximal growth response in the Nb2 cells. Antibodies against the PRL receptors are able to abolish the PRL-induced proliferation of Nb2 cells. In the absence of PRL, these antibodies alone can induce proliferation of Nb2 cells, mimicking the action of PRL. Divalent (Fab)2 fragments, but not monovalent Fab, were also effective. These observations suggest that antibodies to the receptor, or the hormone itself, initiate a biological response possibly by cross-linking PRL receptors on the cell membrane, and that the entry of the PRL molecule, or fragments of it, into the intracellular compartment is not necessary for the biological action of PRL.

Tumors in rats of the Nb strain, arising either spontaneously or after prolonged treatment with s.c. pellets of estrogen, were transplanted to establish whether hormone conditioning was required for their growth. Whereas all spontaneous tumors arising in males and many of those in females were autonomous on transplant, most of those arising in estrogenized rats continued to require hormones for growth after transplantation. The latter included carcinomas of the adrenal cortex, breast, pituitary ectopic tissue, ovary (thecomas), Leydig cells of testis, thymus, pancreas,salivary glands, oribital gland (fibroadenoma), liposarcoma, and lymphoma. Many of the tissues of origin of the tumors have not been considered to be under theinfluence of estrogens. A type of hormone-responsive tumor that was inhibited by estrogen and that grew only in normal rats is described. Ali estrogens tested, including estriol , were interchangeable in action. The incidence of the more common tumors of the adrenal, breast, and pituitary was very low in normal rats, but higher in females. All tumors were more common after estrogenization in both sexes, particularyly in older animals. The secretion of steroids and pitiutary hormones by many tumors led to obvious biological effects. Pituitary secretion led to severe lesions frequently associated with diseases in humans, but the signs of such diseases in the rat apparently were hormone dependent and disappeared if the tumor was removed. The overall results raised the possiblity that estrogens were not carcinogenic per se but stimulated the growth of previously altered cells and that, following their transplantation, this hormone requirement was retained. Irrespective of the mechanism of carcinogenesis, hormone-dependent tumor growth was not irreversible but was controlled in an unexpectedly wide spectrum of organs by exogenous estrogen. Host factors may play a major role in controlling the growth of many tumors and the ultimate course of the disease.

A continuation of previous studies of hormone-dependent tumors in various organs in Nb rats concerns the effects of removal of the hormone stimulus from animals with growing tumors. Tumor regression usually followed this procedure, and in various models it was associated with an increased survival of the animal. A regressed tumor could be caused to grow at any time by estrogen treatment, and the resulting tumor remained hormone dependent, although some progression might occur. Continuous breeding rarely affected the growth or progression of transplanted adrenal or breast carcinomas. When spontaneous regrowth of tumors took place following removal of the estrogen stimulus all types of tumors (except leiomyomas of the uterus, showed progression usually to autonomy, and in the case of male rats bearing breast carcinomas it was inevitable. The substitution of pellets containing a reduced level of estrone to determine which prevented regression and allowed uninterrupted growth offered an assessment of the type or amount of hormone required for the growth of different tumors. By means of such a model of breast cancer in male rats, it was possible to demonstrate that a reduction in hormone levels sufficient to prevent advancing tumor growth, but adequate to reduce the extent of regression, also reduced the frequency or prevented the development of autonomous change. Although regression per se was not a prerequisite for autonomous change, the paradox was evident that progression towards autonomous growth was accelerated with procedures expected to check tumor growth and was minimal with procedures that accelerated it. Liver metastases of hormone-dependent adrenal carcinomas continued growth and could not be influenced by removal of estrogen, although the primary transplant regressed. When such metastases were transplanted, they were not found to have progressed to autonomy but retained a hormone-dependent status. Some tumors, when maintained in estrogen-conditioned hosts, apparently showed a reversion to a more hormone-dependent cell type rather than the expected progression towards autonomy. A theory is suggested to explain the experiments findings on the development and control of estrogen-responsive tumors.

Human GH (hGH) and PRL (hPRL) were iodinated using lactoperoxidase. The iodinated hormones were characterized by RIA, radioreceptor assay (RRA), and bioassay (BA) using the Nb2 Node lymphoma cell line. The proportion of tracer that could bind to rat liver membranes or rabbit antibodies was determined, and the distribution of iodinated hormones was examined using polyacrylamide gel electrophoresis. Excess antibody was capable of precipitating 87.9% of the radioactivity associated with the hGH tracer and 86.0% of the hPRL tracer. The maximal specific binding to a liver membrane preparation averaged 67.3% of the [125I]iodo-hGH radioactivity and 48.8% of the [125I]iodo-hPRL radioactivity. The respective BA and RRA activity estimates for [125I]iodo-hGH averaged 90% and 114% of the activity measured by the RIA. For [125I]iodo-hPRL, the values were 75% by BA and 68% by RRA. The bioactivity profiles of iodinated hGH and hPRL shifted anodally on polyacrylamide gel electrophoresis in comparison to the bioactivity distribution of the respective uniodinated hormones. Iodine incorporation rather than oxidation appeared to be responsible for the shift. After electrophoresis, all eluates which contained significant radioactivity were active in the BA and RIA. Furthermore, specific activities calculated from the bioactive hormone and radioactivity in each electrophoretic segment agreed well with the average specific activity estimated from the amount of iodine incorporated into the protein peak upon gel filtration. These data suggest that hGH and hPRL to a major degree retain biological integrity after iodination.

A study was undertaken to determine if dietary deficiencies of folic acid would influence rotaviral diarrheal disease in infant mice. Female mice were fed diets containing essentially no folic acid, 25% of a normal quantity of folic acid, or a normally recommended quantity of folic acid, beginning at time of breeding and continuing through periods of gestation and lactation. Two-day-old infants from these dams were exposed to purified murine rotavirus or to sterile virus diluent and the severity of the rotaviral infection monitored. Infants from the low folic acid group had significantly lower folate levels in their livers, indicating a deficiency was achieved, and developed more severe disease manifestations than those infants from the dams receiving the normal folic acid levels in their diet. The infection enhancement was seen as increased incidences of diarrhea and a significantly greater number of mice exhibiting high intestinal rotaviral antigen titers. Serum rotavirus antibody titers were below detectable levels in a significant number of these same infants.

Tumor tissue derived from a nephroblastoma arising spontaneously in an Nb hooded rat and carried for 56 generations as a subcutaneous syngeneic transplant was examined by electron microscopy. Histologically, the transplant showed the typical pattern of dense clusters or sheets of basophilic epithelioid cells set within ramifying tracts of fibrous mesenchyme. At the ultrastructural level, the tumor cells within dense clusters were arranged in anastomotic cords that circumscribed clear intervening spaces. The tumor cells were a monomorphic population possessing the morphologic characteristics of blast cells, including polyribosomes as the predominant cytoplasmic organelle. Furthermore, the close contiguity of cells within the cords, their polyhedral form, and their connection by intercellular junctions established the tumor cell type as epithelial. Consistent with benign stroma, mesenchymal tracts contained highly differentiated, mature fibroblasts, collagen, phagocytes, cells of the lymphoid series, and normal blood vessels. No transitional forms between epithelium and mesenchyme suggestive of bipotential differentiation were seen. Although the data represent only a single transplantation line, the observations support the contention (based on light microscopic appraisal of a number of primary tumors) that nephroblastoma in te rat can be a purely epithelial neoplasm.

The growth of a transplantable malignant lymphoma in Nb rats, the Nb 2 node, was accelerated in rats bearing an estrogen pellet and, in particular, in rats bearing transplanted tumors of the anterior pituitary gland. "Lymphoma cell growth-promoting activity" in the sera from the rats was detected and assayed using cultures of Nb 2 node cells. The "activity" of the sera paralleled the degree to which lymphoma growth was accelerated in the animals. Serum from normal rats at a concentration of 1% was moderately active in stimulating culture growth; serum from estrogenized rats was at least 10 times more active; serum from rats bearing pituitary tumors was extremely active and stimulated growth even at a concentration of 0.001% (1:10(5) dilution). In contrast, serum from hypophysectomized rats was devoid of activity, even if the animals were estrogenized. The evidence indicates (a) that the growth of the lymphoma in Nb rats was stimulated by factor(s) in the peripheral blood, the levels of which were subject to control by the pituitary, and (b) that estrogen stimulated lymphoma growth indirectly, through the pituitary gland.

Mammary carcinoma was induced in female Nb rats by implanting pellets of estrone at 25-28 days of age, and prostatic cancer was induced in male rats of the same strain by repeatedly implanting pellets of testosterone propionate. Feeding semipurified diets containing either 3% or 20% corn oil did not significantly affect the incidence or yield of mammary tumors, although tumors tended to appear somewhat earlier in animals on the high-fat diet, confirming the report of Dunning et al. published nearly 40 years ago. The yield of prostatic tumors was too low to draw any conclusions with respect to dietary fat. Hormonally induced mammary tumors appear to be less responsive to dietary fat than those induced by commonly used chemical carcinogens.

The inactivation (desensitization) of the acetylcholine receptor by carbamylcholine, a stable analogue of acetylcholine, has been investigated in eel Ringer's solution, pH 7.0, 0 degrees C, by measurements of (i) ion flux and (ii) the kinetics of the reaction of [125I]-alpha-bungarotoxin with the receptor. The effect of preincubation with carbamylcholine is significantly different in the two types of measurement. In both the receptor-controlled flux of inorganic ions and the toxin-binding kinetics a biphasic process has been observed (Hess, G.P., Lipkowitz, S., Struve, G.E., 1978, Proc. Nat. Acad. Sci. USA 75:1703; Hess, G.P. et al., 1975, Biochem. Biophys. Res. Commun. 64:1018; Bulger, J.E. et al., 1977, Biochemistry 16:684), only the initial fast phase of which is inhibited and the subsequent slow phase persists. However, preincubation with carbamylcholine per se has no effect on the toxin reaction. The results obtained are consistent with the proposal of Katz and Thesleff (Katz, B., Thesleff, S., 1957, J. Physiol. (London) 138:65) that the active form of the receptor is converted to an inactive form in the presence of acetylcholine receptor ligands, and with our previous experiments (Hess et al., 1978) which indicated that one receptor form is responsible for the initial fast phase of both the receptor-controlled ion flux and the toxin binding reaction, and that its conversion to the other form results in the slow phases in these two measurements.

The amino terminal 54 residue peptide fragment of human somatotropin [Cys(Gam)53]-HGH-(1-54), has been synthesized by the solid-phase method. The symmetrical anhydride and active ester coupling methods were used exclusively. The synthetic product was purified by gel fitration isoelectric focusing, and partition chromatography. It was found to be homogeneous by six additional criteria. In complement fixation experiments the synthetic product was immunologically active with antisera to HGH and [cys(Cam)53]-HGH-(134). Antiserum raised against the synthetic product was immunologically active in the homologous assay and with HGH,[Cys(Cam)53]-HGH-(1-134), and [Cys(Cam)53]-HGH-(15-125). The synthetic fragment exhibited 53% of the activity of [Cys(Cam)53]-HGH-(1-134) in the rat tibia assay.

A malignant Nb rat lymphoma which in vivo is stimulated by estrogens has been established in suspension culture. The cultured cells grew readily in Fischer's medium supplemented with fetal calf serum (10%) and 2-mercaptoethanol (10(-4) M). If horse serum was substituted for fetal calf serum, population growth ceased; i.e., cultures became "stationary." Such stationary cultures could be induced to resume active growth by the addition of a pituitary hormone, prolactin (ovine, rat); concentrations as low as 10 pg/ml had a detectable effect. In contrast, other pituitary hormones or estrogens had little or no effect. The evidence in this and an accompanying paper suggests that prolactin (or related substances) has a role in the growth of some cancers of lymphoid origin in rats.

Comparisons are described between Bungarus caeruleus venom and the actual venom from which a putative marker for the cholinergic ionophore, called ceruleotoxin, was isolated. The venoms are shown to be different by two procedures for ion exchange chromatography and by isoelectric focusing on polyacrylamide gel. The activities of the purified "ceruleotoxin" as an inhibitor of acetylcholine receptor-mediated ion flux and as a phospholipase have been reported (Bon & Changeux, 1977b). The results reported herein suggest that this toxin is from an unknown origin.

Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, u.v. spectroscopy, and analytical high performance liquid chromatography.

Androgen-stimulated tumors of the dorsal prostate were derived from an estrogen-dependent tumor of Nb rats (Nb-2Pr-E). The change followed repeated androgen treatments in a rat bearing a tumor which had regressed after estrogen removal. The eventual tumor regrowth provided tumor transplant lines, Nb-2Pr-A, which grew in normal or androgenized males, slowly in castrates, but not in estrogenized females. The Nb-2Pr-A growing tumors regressed after treatment with estrogen, and tumors which eventually regrew were autonomous. Slow tumor growth in castrated animals also responded to estrogen treatment. Tamoxifen, an antiestrogen, caused tumors growing in males to become stationary or regress. Regrowth of tumors after they had responded to tamoxifen again responded to castration or estrogen. Tumors which escaped from estrogen treatment or were autonomous did not respond to tamoxifen. The most effective sequence for treatment was tamoxifen followed by castration and, finally, estrogen. Tamoxifen treatment or castration allowed tumors to remain hormone responsive, whereas initial estrogen treatment prevented response to subsequent therapy and led to an autonomous change.