Showing papers by "Worcester Foundation for Biomedical Research published in 1982"

Purification and polypeptide composition of dynein ATPases from Chlamydomonas flagella.

Abstract: Extraction of isolated, demembranated flagellar axonemes of Chlamydomonas reinhardii with 0.6 M KCl solubilized 77-92% of the total axonemal Mg++ or Ca++-ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPases of these two peaks were further purified by hydroxyapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of approximately 2.0 +/- 0.5 mumoles Pi hydrolyzed min/mg and was associated with four high molecular weight (HMW) polypeptides of approximately 310,000-340,000 daltons, two intermediate molecular weight (IMW) polypeptides of 78,000 and 69,000 daltons, and eight low molecular weight (LMW) polypeptides of 7,800-19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in two peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of approximately 0.6 +/- 0.3 mumoles Pi hydrolyzed min/mg and associated with approximately 330,000-, 21,700-, and 18,100-dalton polypeptides. The 10.5S peak contained several high, intermediate, and low molecular weight polypeptides; of these, one HMW polypeptide and one 28,700-dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents approximately 9.2% of the total axonemal protein; 12S dynein represents approximately 4.7% of the axonemal protein.

Metabolism of round spermatids from rats: lactate as the preferred substrate

Abstract: Round spermatids were prepared from rat testes and incubated with various substrates (glucose, fructose, pyruvate, lactate and acetate) to measure utilization of substrates and production of ATP in the presence of saturating levels of each substrate. By both criteria lactate is the preferred substrate by a factor of 3 or 4. Production of more than half of the ATP with lactate is substrate is prevented by addition of an inhibitor of alpha-ketoacid dehydrogenase (5-methoxyindole-2-carboxylic acid) Pyruvate and lactate are interconverted and pyruvate inhibits production of ATP from lactate. Synthesis of ATP with lactate and with pyruvate is inhibited by rotenone, rutamycin or 2,4-dinitrophenol. Utilization of glucose is limited by aldolase activity. These findings suggest that exogenous lactate is oxidized by lactate dehydrogenase followed by pyruvate dehydrogenase and Krebs; cycle enzymes under conditions which do not allow pyruvate to inhibit lactate dehydrogenase. ATP is synthesized through electron transport. Post-mitochondrial supernate from spermatids showed that high concentration of pyruvate (greater than 1 mM) inhibit lactate dehydrogenase with pyruvate as substrate and that with lactate as substrate, pyruvate behaves as a competitive inhibitor of lactate dehydrogenase. Evidently lactate is the preferred substrate for round spermatids and energy production is most efficient when this substance is present in high concentrations and pyruvate is present in low concentrations. Reasons are given for suggesting that Sertoli cells may provide the relatively large amounts of lactate required by round spermatids.

Laminar organization of mitral and tufted cells in the main olfactory bulb of the adult hamster.

Abstract: Morphometric analyses of Golgi-impregnated mitral and tufted cells in the main olfactory bulb of the hamster indicate that these second-order neurons cannot be considered a homogeneous population. They are comprised of several categories based on differences in the sizes and spatial organizations of their dendritic fields and in their likely patterns of lateral and recurrent interactions with granule cells and/or periglomerular cells. External tufted cells have their somata in the periglomerular region or at the superficial border of the external plexiform layer (EPL) and can be subdivided into three morphological categories. Many external tufted cells lack secondary dendrites, i.e., all of their dendrites arborize within glomeruli. Most of these neurons are not in positions where they could receive synaptic contacts from granule cells and thus are likely to receive interneuronal input predominantly from periglomerular cells. Another category of external tufted cells is characterized by secondary dendrites which are highly branched and form dense but spatially restricted fields in the EPL. In contrast, external tufted cells in the third category have sparsely branched secondary dendrites which extend tangentially in the EPL and tend to be asymmetrically distributed with respect to the soma. Most of the deeperlying tufted cells, and the mitral cells, have sparsely branched secondary dendrites whose lengths and laminar distributions within the EPL are correlated with the depths of their parent somata. Internal tufted cells, which have their somata in the deep one-third of the EPL, and mitral cells exhibit the largest secondary dendritic fields. These fields extend tangentially within the EPL in relatively symmetric radial patterns from their parent somata and envelop substantial proportions of the EPL. Middle tufted cells have their somata in the superficial two-thirds of the EPL and exhibit secondary dendritic morphologies which are intermediate between those of internal and external tufted cells. These differences in the size and spatial organization of secondary dendritic fields are likely to be associated with differences in the amount and spatial distribution of lateral and recurrent inhibition mediated through synaptic contacts with granule cells. External tufted cells which lack secondary dendrites have large glomerular arbors that are comparable in size to those of mitral and internal tufted cells, whereas external tufted cells with secondary dendrites and middle tufted cells tend to have relatively small glomerular arbors. The morphological differences among mitral and tufted cells and the likely differences in their synaptic interactions within the olfactory bulb, together with recent evidence that these neurons differ in their pharmacology and in their patterns of interconnections with more central regions of the forebrain, suggest that the mitral and tufted cells and the central olfactory circuits may be organized as functionally defined parallel pathways.

Localization of methionine‐enkephalin, substance P, and somatostatin immunoreactivities in the main olfactory bulb of the hamster

Abstract: Methionine-enkephalin, substance P, and somatostatin immunoreactivities are associated with different classes of neurons in the main olfactory bulb of the hamster. In the glomerular layer, methionine-enkephalin immunoreactivity is observed in the somata and dendrites of periglomerular cells whereas substance P immunoreactivity is present in the somata and dendrites of external tufted cells. Substance P immunoreactivity may also be present in the superficial short axon cells. In the deeper layers, methionine-enkephalin immunoreactivity is observed in a mixed population of neurons which appear to be primarily granule cells, whereas somatostatin immunoreactivity is present only in deep short axon cells. Methionine-enkephalin-, substance P-, and somatostatin-positive fibers of central origin are also observed in the main olfactory bulb.

Stimulation by follicle-stimulating hormone of synthesis of lactate by Sertoli cells from rat testis.

Abstract: Sertoli cells from rats aged 16 days were cultured in defined medium for 2 days and then treated by addition of fresh medium containing various hormones (treated) or saline (control). The concentration of lactate in the medium was measured as a function of time. The production of lactate measured under these conditions was increased by addition of FSH to the medium. For NIH FSH (13 and 14), ED50 for stimulation of lactate production was approximately 0.05 micrograms/ml. Stimulation was also seen with LH and TSH (ED50, 0.8 micrograms/ml for both hormones). Reasons are given for believing that TSH may possess the inherent capacity to stimulate production of lactate in contrast to LH, which appears to act only by way of contaminating traces of FSH. Dibutyryl cAMP also stimulates lactate production by Sertoli cells. Other hormones tested, including androgens, were without effect. When production of lactate by Sertoli cells was compared with the maximal consumption by spermatids in vitro, it became apparent that Sertoli cells may provide the major source of metabolic substrate for spermatids in the form of lactate. Stimulation of lactate production by FSH was inhibited by puromycin, cycloheximide, and actinomycin D. Evidently this response requires synthesis of new protein and RNA. This effect of FSH may be important in the regulation of spermatogenesis.

Alterations in neuroendocrine function during photoperiod induced testicular atrophy and recrudescence in the golden hamster.

Abstract: Transfer of golden (Syrian) hamsters from a 14L:1OD (light:dark) to a 5L:19D photoperiod causes profound changesinthe reproductive axis. Testicular weight fell within 7 weeks of short photoperiod exposure and remained low until 19 weeks, at which time spontaneous recrudescence began. Plasma LH, FSH and PrI levels were undetectable at 7.5 weeks of exposure, but LH and FSH returned to basal levels by 15 weeks and PrI by 20.5 weeks. Hypothalamic LHRH levels increased concurrently with the fall in LH and FSH, suggesting that LHRH release was decreased. Increasing plasma gonadotropin levels and testicular recrudescence were associated with decreases in hypothalamic LHRH content. Hypothalamic norepinephrine (NE) turnover was low at 10 weeks, suggesting that decreased LH and FSH levels were ultimately due to decreased adrenergic activity. At 15 weeks, plasma LH levels and hypothalamic NE turnover were both increased over values seen at 10 weeks. Hypothalamic dopamine turnover, possibly reflecting decreased PrI feedback, was decreased between 0 and 10 weeks, but returned to control levels by 15 weeks. It, therefore, appears that the endocrine changes associated with photoperiod induced testicular regression and recrudescence are secondary to changes in hypothalamic function. BIOLOGY OF REPRODUCTION 26,437-444 (1982)

Complete nucleotide sequence of a sea urchin actin gene

Abstract: We have determined the complete nucleotide sequence of a sea urchin actin gene, including the entire protein-coding sequence, introns and approximately 500 and 700 nucleotides adjacent to protein-coding-sequence on the 5' and 3' sides, respectively. This gene is split between codons 121 and 122 and within codon 204 by two introns which are 233 and 181 nucleotides in length, respectively. Comparison of the sequence of the two introns indicates a region of distant relatedness which covers about 25% of their lengths, suggesting that these sequences might have derived from a common ancestral sequence. The encoded amino acid sequence, which matches plasmic-like than muscle-like when compared to vertebrate actins. Analysis of the coding-flanking regions indicates the presence of sequences similar to those thought to be important for initiation of transcription and polyadenylation of mRNA. The location of these sequences and the size of an actin mRNA, transcribed from this or a very closely related gene, suggests that initiation occurs 347 nucleotides 5' of coding and polyadenylation approximately 515 nucleotides 3' of coding.

Etiology of DES-Induced Uterine Tumors in the Syrian Hamster

Abstract: This paper describes a new experimental model system for the induction of endometrial adenocarcinoma in hamster uterus following diethylstilbestrol (DES) treatment of the newborn female. We propose that DES acts as an initiator during early development and that other estrogens act as promoters to stimulate tumor development in the adult uterus. DES directly affects the uterus as was shown by the failure of neonatal ovariectomy to prevent early DES-induced uterine growth. Subsequently, ovarian estrogen secretion from anovulatory, polyfollicular ovaries modifies the DES-altered uterus starting between 20 and 30 days of age and continuing into adult life. Early DES effects on the uterus include stimulation of endometrial cellular differentiation and progesterone receptor production. Permanent changes in uterine collagen, DNA and progesterone receptor content were noted, but the responsiveness of the DES-altered uterus to estrogen and progestin action was not impaired. Morphogenetic changes included an increase in extracellular connective tissue elements and striking alterations in endometrial cell composition such as hyperplasia of luminal and glandular epithelia and a massive inflammatory response in the stroma. Endometrial adenocarcinomas occurred in DES-treated animals in association with exposure to either endogenous estrogen from anovulatory ovaries or exogenous estrogen treatment of the ovariectomized animal. Endometrial tumors had relatively high concentrations of estrogen and progesterone receptors, suggesting a sensitivity to hormone action. Thus, these studies (a) demonstrate the utility of this animal model for the preparation of experimental endometrial tumors, and (b) suggest that DES acts as an initiator to transform uterine cells during early development, and estrogen exposure later in life acts as a promoter to stimulate growth and proliferation of DES-transformed cells.

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA.

Abstract: Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.

A microtubule-associated protein in the mitotic spindle and the interphase nucleus

Abstract: Accurate chromosome segregation during mitosis is contingent on the controlled polymerization and function of spindle microtubules. Assembly-promoting polypeptides that co-sediment with microtubules during cycles of polymerization in vitro are attractive candidates for regulators of the formation and function of the mitotic apparatus in vivo. However, previous work has shown that the major species of microtubule-associated proteins (MAPs) from mammalian brain or tissue culture cells are either not associated with the mitotic spindle1 or are associated with both mitotic and interphase cytoplasmic microtubules2–4. We have therefore sought a novel way to identify spindle regulatory factors. In the study reported here, monoclonal antibodies have been prepared against a MAP fraction from HeLa cells. One of the resulting hybridoma clones produces IgG that binds to a polypeptide of molecular weight ∼200,000. During cell division this antigen is associated with fibres in the mitotic apparatus, but in interphase it is located primarily in the nucleus. Surprisingly, the antigen is also detectable on the plasma membrane of circulating human erythrocytes. This spindle component is distinct from previously characterized MAPs and may represent a regulatory factor in the assembly and function of the mitotic apparatus.

Stimulant actions of histamine H1 antagonists on operant behavior in the squirrel monkey.

Abstract: Squirrel monkeys were studied under fixed-interval schedules of reinforcement in which the first response (lever press) after a fixed period of time resulted either in the delivery of a food pellet or in the termination of stimuli associated with impending electric shock delivery. Benztropine mesylate (0.03–1.7 mg/kg), promethazine HCl (0.3–10 mg/kg), and diphenhydramine HCl (0.3–17 mg/kg) all produced marked increases in responding at intermediate doses. The increases in responding were at least as great as those observed with psychomotor stimulants, such as amphetamine, in this species under similar behavioral conditions. Benztropine was most potent and diphenhydramine was least potent in most monkeys and, in some, promethazine and diphenhydramine were about equipotent. The order of potency and the magnitude of potency differences among the drugs suggest that the behavioral effects were due to antagonist actions at histamine H1 receptors, rather than to effects on dopamine uptake or on muscarinic receptors.

Most of the Murine Leukemia Virus Sequences in the DNA of NIH/Swiss Mice Consist of Two Closely Related Proviruses, Each Repeated Several Times

Abstract: The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol. 43:113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.

Inhibition of Estrogen Biosynthesis and Regression of Mammary Tumors by Aromatase Inhibitors

Abstract: Aromatase inhibitors are potentially useful therapeutic agents for patients with estrogen-dependent cancer. The rationale for this approach is that compounds interacting with aromatizing enzyme in all estrogen producing tissues could provide both selective and effective inhibition. The conversion of androgens to estrogens is a unique reaction in the biosynthesis of steroids since it involves aromatization of the A ring of the steroid molecule. This is the last series of steps in the biosynthetic progression from cholesterol to the estrogens. Therefore, compounds inhibiting the enzyme system mediating aromatization would be expected to be more specific inhibitors of estrogen biosynthesis than those influencing steps earlier in steroidogenesis (Lipton and Santen, 1974; Levin et al., 1976). In women, estrogens are produced primarily by the ovaries but extragonadal tissues (Longcope et al., 1978; Hemsell et al., 1974) such as fat, muscle and certain breast tumors (Miller et al., 1974; de Thibault et al., 1974; Valera and Dao, 1978) also synthesize estrogens which contribute to tumor growth. Thus, aromatase inhibitors acting in all tissue sites may be more effective as well as more specific than present methods for reducing estrogen production.

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerme alpha and bacterial DNA polymerase III

Abstract: The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Effects of dopamine uptake inhibitors on schedule-controlled behavior in the squirrel monkey.

Abstract: Squirrel monkeys responded under a multiple fixedinterval (FI) fixed-ratio (FR) schedule of stimulus-shock termination. Benztropine mesylate (0.03–1.7 mg/kg), bupropion HCl (0.3–5.6 mg/kg), mazindol (0.01–0.3 mg/kg), and nomifensine maleate (0.1–1.0 mg/kg) markedly increased responding under the FI schedule, but not under the RR schedule. Mazindol was about three-times more potent than nomifensine and ten-times more potent than bupropion. Benztropine and mazindol were about equal in potency. The order and relative magnitude of potency differences for mazindol, nomifensine, and bupropion are similar to those reported by others for in vitro inhibition of dopamine uptake in rat striatum, but the relative potency of benztropine was greater in these behavioral experiments than expected from its potency in inhibiting dopamine uptake.

Mitosis, cytokinesis and colony formation in the colonial green alga Astrephomene gubernaculifera

Abstract: Interphase cells of Astrephomene resemble those of other volvocalean algae, except for the insertion of nearly parallel flagella into a depression or shallow pit. During prophase, virus-like particles become associated with the condensed chromatin where they remain throughout mitosis. Except for polar fenestrae, mitosis is closed. Two centrioles are located laterally to each pole of the curved spindle. Microtubules insert into the chromosomes at three layered kinetochores. Spindle microtubules remain surrounded by the nuclear envelope through telophase. Phycoplast microtubules radiating from each pair of centrioles predict the plane of cytokinesis. After several divisions a curved plate of cells develops with the centrioles of each cell toward the convex surface. Additional divisions produce a hollow sphere but, unlike members of the Volvocaceae, no inversion occurs. Cytoplasmic bridges, resembling similar structures in the Volvocaceae, are formed by incomplete cytokinesis. Since the bridges in the Volvoc...

Effects of Experimentally Induced Hyperprolactinemia on the Hypothalamus, Pituitary, and Testes in the Golden Hamster

Abstract: Hyperprolactinemia was induced in adult male golden hamsters by transplantation of four homologous pituitaries under the renal capsules. The resulting elevation of plasma prolactin (PRL) levels was accompanied by a dramatic increase in the concentration of FSH in peripheral plasma. In contrast, plasma LH levels were not affected. The content and concentration of testicular LH receptors, testicular weight, and plasma testosterone levels were significantly greater in hyperprolactinemic hamsters than in the sham-operated control animals. Experimentally induced hyperprolactinemia was associated also with an increase in the hypothalamic content of LHRH and norepinephrine, while the dopamine level in the hypothalamus was not affected. Furthermore, hyperprolactinemia appeared to have increased the release of LH and FSH from the incubated hemipituitaries in the presence and in the absence of LHRH, but only release of LH under basal conditions was significantly affected. The authors conclude from these observations that experimentally induced hyperprolactinemia stimulates testicular function in the golden hamster by increasing the release of endogenous FSH and by increasing the number of LH receptors in the testes. Comparison of these findings with the results of similar experiments in rats and mice suggests that the number of testicular LH receptors can be profoundly influenced by chronic changes in plasma levels of both LH and PRL and that PRL can stimulate LH binding only when LH levels are reduced or unaltered.

Effects of alcohols upon pacemaker activity in neurons of Aplysia californica

Abstract: 1. Alcohols have been used as pharmacological tools to probe the nature of action-potential gates and channels. In this study, we examine the effects of alcohols upon activity patterns in Aplysia neurons. 2. Ethanol at concentrations of 0.4-0.6 M induces bursting pacemaker activity (BPA) in previously silent cells. The same effect is produced with 40-60 mM concentrations of butanol, suggesting that this induction is not due to osmotic effects. 3. Voltage-clamp measurements indicate that the induction of BPA is accompanied by the appearance of a negative-slope resistance (NSR) region in the steady-state current-voltage relationship of the cell. The induction of BPA and a NSR region in silent cells is antagonized by lowered temperatures. 4. Ethanol concentrations which produce BPA and a NSR region in silent cells abolish both of these normally present characteristics in endogenous bursters. This suggests that whatever membrane components are moved into optimal configuration for the expression of BPA in silent cells are shifted out of optimal configuration in endogenous bursters, by similar ethanol concentrations.

The presence of 17β-hydroxysteroid dehydrogenase activity in preimplantation rat and mouse blastocysts

Abstract: When Day 5 rat blastocysts and Day 4 and 5 mouse blastocysts were cultured in 53 microliters of medium containing 1340 or 2680 pg [3H]estradiol (E2), large amounts of [3H]estrone (E1) were detected in the medium at daily intervals for up to 5 days. This indicates the presence of 17 beta-hydroxysteroid dehydrogenase in the embryos. The activity was higher at a higher concentration of E2 and was also higher in mouse than in rat blastocysts. In the mouse, the activity was higher in Day 5 than Day 4 blastocysts during the first day in culture; then it decreased in Day 5 but increased in Day 4 blastocysts. The importance of E2 in embryonic development and implantation as suggested by others may be related to the activity of 17 beta-hydroxysteroid dehydrogenase.