Showing papers by "Worcester Foundation for Biomedical Research published in 1986"

A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family

Abstract: A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000 The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus

Product of per locus of Drosophila shares homology with proteoglycans

Abstract: Genes controlling biological rhythms have been identified in Drosophila. The best characterized of these genes is called period (per). Although wild-type flies have daily (circadian) rhythms with a periodicity of approximately 24 h, pers and per1 mutants have 19-h and 29-h rhythms, respectively, and pero mutants are arrhythmic. The pers mutation also enhances the sensitivity of the circadian clock to resetting by light stimuli, and all three types of per mutations affect a much shorter period ultradian rhythm, the 55-s rhythm of the Drosophila courtship song. A fragment of DNA of approximately 7 kilobases (kb) encoding a 4.5-kb poly(A)+ RNA restores rhythmicity when transduced into Drosophila carrying mutations or chromosomal deletions of the per locus. Here we report the sequence of this biologically active segment of DNA. The transcription unit that encodes the 4.5-kb RNA has been mapped, permitting a conceptual translation of a protein of 1,127 amino acids. Several abnormal phenotypes characterized by long-period rhythms are associated with changes in the sequence of untranslated portions of the transcription unit. The structure of some segments of the predicted protein suggests that it is a proteoglycan.

Maternal transfer of photoperiodic information influences the photoperiodic response of prepubertal Djungarian hamsters (Phodopus sungorus sungorus).

Abstract: Daylengths during the spring are repeated in reverse order in the autumn. For some photoperiodic species, a given photo period may be stimulatory for reproduction in the spring and inhibitory in the autumn. The mechanisms regulating this type of seasonal response have, until recently, remained a mystery. Horton (1984a) showed in Microtus montanus that the photoperiod experienced by the mother influences the gonadal development of her young after weaning. To determine if this phenomenon is characteristic of other photoperiodic rodents, adult Djungarian hamsters were paired on 16L:8D, 14L:1OD, or 12L:12D. Young males born from these pairings were killed at 15, 28, and 34 days of age to assess gonadal development (testes weight). At 15 days testicular development was identical in all groups; by 28 days, however, males raised in 16L:8D or 14L:1OD exhibited a greater degree of testicular development than those raised in 12L: 12D. Next, females maintained on each of the three photoperiods throughout gestation were transferred, with their offspring, to the other two photoperiods at birth. Postnatal exposure to 14L:1OD or 12L:12D inhibited testicular development in young that had been gestated on 16L:8D. Both 16L:8D and 14L:1OD stimulated testicular growth in animals that had been gestated on 12L: 12D or 14L: IOD. Therefore, a) 16L:8D stimulates testicular growth in all animals, b) 12L:12D inhibits testicular growth in all animals, and c) the testicular response to 14L:IOD depends on the photoperiod experienced by the mother during pregnancy. The fact that maternal transfer of photoperiodic information influences prepubertal photoperiodic responses could have important implications for sexual development in animals born in late spring/early summer as compared to those born in late summer/early fall.

Circadian regulation of pineal melatonin and reproduction in the Djungarian hamster.

Abstract: Gonadal state, pineal melatonin rhythms, and locomotor activity rhythms were examined in juvenile male Djungarian hamsters exposed to non-24-hr light cycles ("T-cycles") or to full photoperiods. At the end of 1 month, hamsters exposed to a 1-hr pulse of light every 24.33 hr (T 24.33) exhibited small testes, whereas those receiving the same amount of light every 24.78 hr (T 24.78) displayed stimulated gonads, ten-fold larger in size. Accompanying the nonstimulatory effect of the T 24.33 cycle were nocturnal peaks in both pineal melatonin content and serum melatonin concentration which were longer by approximately 4 hr than those observed on the photostimulatory T 24.78 cycle. Exposure to an intermediate-length T-cycle (T 24.53) resulted in a mixed gonadal response and in pineal and serum melatonin peaks of intermediate duration. Wheel-running activity was entrained to the T-cycles such that light was present only near the beginning of the subjective night, its phase (relative to activity onset) differing only slightly among T-cycle groups. Hence the durational differences observed in the melatonin peaks were apparently not due to the acute suppressive or phase-advancing effects of morning light on melatonin biosynthesis, but were rather the result of differences in the endogenous control of pineal activity by the circadian pacemaker system. While no strong correlation was detected between gonadal state and the phase of locomotor activity onset relative to the light pulse, a significant correlation was observed between gonadal state and the duration of daily locomotor activity (alpha). These data were compared to similar measures obtained from hamsters exposed to long-versus short-day full photoperiods (LD 16:8 vs. LD 10:14). In summary, the results of this study indicate involvement of the circadian pacemaker system of Djungarian hamsters in the control of pineal melatonin synthesis and secretion, and in photoperiodic time measurement. Furthermore, these data strengthen the hypothesis that it is the duration of nocturnal pineal melatonin secretion that is the critical feature of this neuroendocrine gland's photoperiodic signal.

Spatial cue utilization in chronically malnourished rats: task-specific learning deficits.

Abstract: Rats whose mothers were maintained on either a 25% casein diet or an 8% casein diet and who were provided the same diet after weaning were tested on delayed spatial alternation or on one of a series of spatial localization problems using the Morris maze (Morris, 1981). Malnourished rats demonstrated perseverative deficits in the form of strings of consecutive errors on the delayed spatial alternation. Performance in the Morris maze indicated spatial localization ability and spatial memory processes were not impaired by chronic malnutrition in rats. The data suggest that complex processing of spatial information that includes flexible use of place cues over short intervals is impaired by malnutrition, while spatial localization per se and spatial mapping are not affected.

The proenkephalin gene is widely expressed within the male and female reproductive systems of the rat and hamster.

Abstract: The distribution of proenkephalin mRNA in reproductive tissues of the rat and hamster was examined by Northern analysis and RNA blot hybridization. In the male reproductive tract of both species, proenkephalin mRNA was detected in the testis, vas deferens, epididyrhis, seminal vesicles, and prostate. Except in the case of the rat testis, the size of this transcript in each organ was identical to that generally observed in tissues expressing proenkephalin (1450 nucleotides). The major proenkephalin mRNA species expressed in the rat testis migrates as a 1900–nucleotide RNA on denaturing agarose gels. In the male rat and hamster, the highest abundance of proenkephalin mRNA was found in the testis and epididymis. A 1450–nucleotide proenkephalin mRNA also was detected in each of the 3 female reproductive tissues of the rat and hamster that were examined: uterus, oviduct, and ovary. These unusual observations suggest that proenkephalin-derived peptides are synthesized at multiple sites within the male and femal...

Single channels and ionic currents in peptidergic nerve terminals.

Abstract: Control of secretion, by a mechanism in which membrane depolarization leads to Ca2+ entry1, has been extensively studied. The small size and inaccessibility of most nerve terminals, however, have precluded direct analysis of membrane ionic currents and their influence on secretion (with some notable exceptions2,3). Recently, patch-clamp methods have been applied to several secretory systems4–6 for both voltage-clamp and single-channel recordings. We now report the extension of this analysis to isolated peptidergic nerve terminals. We used terminals obtained from a crustacean neurohaemal organ, the sinus gland. Analyses of currents under whole-terminal voltage clamp showed inward currents carried by Na+ and by Ca2+, and outward currents carried predominantly by K+. Furthermore, we have observed two types of single-channel currents that may be unique to nerve terminals. Both show little selectivity between Na+ and K+. The first channel is activated by intracellular Na+ and the second by intracellular Ca2+. These channels have conductances of 69 and 213 pS, respectively, in symmetrical 310 mM KCl. It should now be possible to compare electrical activity recorded intracellularly from intact nerve endings7,8, with whole-cell and single-channel currents and with the release of peptide neurohormones from isolated neuronal terminals.

The tip-E Mutation of Drosophila Decreases Saxitoxin Binding and Interacts with Other Mutations Affecting Nerve Membrane Excitability

Abstract: A recessive temperature-sensitive paralytic mutation, tip-E, is associated with reduced binding of [3H]saxitoxin to voltage-sensitive sodium channels in membranes from adult Drosophila heads. There is a decrease of 30-40% in the number of [3H]saxitoxin-binding sites per mg protein (Bmax), but the dissociation constant (Kd) for [3H]saxitoxin binding is normal in the remaining population of binding sites. This decrease is not due to a general hypotrophy of neural tissue since the number of alpha-bungarotoxin binding sites is normal in tip-E mutants. Although saxitoxin binding is reduced in vitro, pharmacological experiments suggest that tip-E mutants have close to the wild-type number of sodium channels in vivo. This suggestion is supported by the observation that at permissive temperatures tip-E only marginally suppresses a mutation which causes enhanced membrane excitability. However, even at permissive temperatures tip-E interacts synergistically with mutations that decrease membrane excitability. In this case, the double mutants exhibit reduced viability and/or longevity. We postulate that either the structure of sodium channels or their microenvironment is altered in tip-E mutants resulting in an increased liability of binding sites in vitro.

Reproductive effects of olfactory bulbectomy in the Syrian hamster.

Abstract: The effects of olfactory bulbectomy on circulating gonadotropin, prolactin and testosterone levels and on the testicular and pituitary responses to shortening of day length were studied in Syrian hamsters. Adult animals maintained on a 14L:10D cycle were sham-operated or sustained bilateral radical olfactory bulbectomies by aspiration to remove the main and accessory olfactory bulbs and the adjacent regions of the anterior olfactory nucleus. They were then maintained either on the long photoperiod or housed on a 10L:14D cycle. Testicular length was measured at weekly intervals over a 5-mo period. Sham-operated controls exhibited the normal pattern of testicular regression and eventual recrudescence on the short photoperiod. Testicular regression was significantly reduced in bulbectomized animals. Many of these animals showed no regression; others exhibited a reduced degree and/or shortened duration of regression. Serum levels of follicle-stimulating hormone (FSH) were substantially elevated in bulbectomized males maintained in long days. Their serum levels of luteinizing hormone (LH), prolactin and testosterone remained within the range for shams on long photoperiod. In short days, the bulbectomized animals showed the normal, pronounced decline in circulating prolactin levels. Serum FSH and LH levels also showed substantial declines, but the FSH levels were not reduced below the range for controls in long days, and the decline in LH levels was not as great as that for controls in short days.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronically administered d-amphetamine on spaced responding maintained under multiple and single-component schedules.

Abstract: Key pressing by rats was maintained under spaced-responding and random-ratio schedules of food delivery, and rates of responding were reliably different for each schedule. When responding was maintained under a multiple schedule, appropriate doses of d-amphetamine (1.0 and 1.7 mg/kg) markedly increased low rates of spaced responding while markedly decreasing high rates of ratio responding. These drug-produced changes in response rate resulted in decreased food presentation during both schedule components. When 1.0 mg/kg d-amphetamine was given daily, tolerance developed to initially decreased ratio responding in six to nine sessions, but did not develop to initially increased spaced responding. However, when the ratio schedule was removed, tolerance developed very quickly to increases under the spaced-responding schedule, and associated food frequency returned to control levels. When the ratio schedule was reinstated, spaced responding was once again increased, and its associated frequency of food delivery was again decreased. The development of tolerance to the behavioral effects of d-amphetamine was influenced more by global changes in response consequences during entire experimental sessions than by local changes in response consequences in single components of those sessions. Whenever the concept of “response cost” is used, it should be understood in terms of total “cost.”

Pineal melatonin rhythms in female Turkish hamsters: effects of photoperiod and hibernation.

Abstract: Daily rhythms of pineal and serum melatonin content were characterized for adult female Turkish hamsters (Mesocricetus brandti) exposed to long days (16L:8D, 22#{176}C) or after transfer to short days (IOL:14D, 22#{176}C). The nocturnal peak of pineal melatonin content was found to be approximately 3 h greater in duration on short than on long days. Changes in levels of serum melatonin closely paralleled those of pineal melatonin. Thus, an effect of photoperiod on synthesis and secretion of pineal melatonin was demonstrated. In a separate experiment, female hamsters were induced to hibernate by exposure to a short-day, cold environment (1OL: 14D, 6#{176}C). During the 4 to 5-mo hibernation season, Turkish hamsters are known to display 4 to 8-day bouts of torpor (body temperature = 7-9#{176}C) alternating with 1 to 3-day intervals of euthermia (body temperature = 3537#{176}C). Little evidence of nocturnal synthesis or secretion of pineal melatonin was detected in females sampled during torpor. However, animals sampled during the first day after arousal from a torpor bout displayed melatonin rhythms no different in phase or amplitude from those seen in females held at 22#{176} C. Thus, despite the absence of pineal melatonin output during torpor, the pineal gland of hibernating Turkish hamsters produces an appropriately phased, rhythmic melato nm signal during intervals of euthermia.

ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100.

Abstract: It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in spermatozoa on leaving the testis, but some fine adjustment must take place during maturation in the epididymis.

Production of components of extracellular matrix by cultured rat Sertoli cells.

Abstract: Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.

Ethanol effects on voltage-dependent membrane conductances: comparative sensitivity of channel populations in Aplysia neurons

Abstract: 1. The study of ethanol (EtOH) action is interesting because of its clinical relevance and for the insights it provides into structure-function relationships of excitable membranes. This paper describes the concentration dependencies of various parameters of four currents inAplysia cells. 2. ICa is the most sensitive of the currents studied. There was a significant reduction ofICa at concentrations of 50 mM EtOH. At low concentrations, the reduction of amplitude was the primary effect of ethanol, with the kinetics and voltage dependency of activation not affected. 3. INa andIA were also affected, but at EtOH levels higher than those which alteredICa. The primary effect of EtOH onINa was a reduction in its amplitude, although the time to peak current flow was increased by EtOH. The effects of EtOH onIA were cell specific and, for the purposes of this paper, we examined the giant metacerebral cell (MCC). In MCC, the primary effect of EtOH onIA was an increase in the time course of inactivation. The time to peakIA was also increased by high concentrations of EtOH, but its amplitude was unaffected even at high concentrations. The delayed rectifier current,IK, was the most EtOH resistant of the currents examined. High EtOH concentrations augmented the amplitude ofIK, although even at 600 mM concentrations, the percentage change was only 30%. 4. Our results indicate that the calcium channel is very susceptible to the influence of ethanol and is a serious candidate to be the primary target of EtOH action in the nervous system. 5. The differential sensitivity of voltage-dependent currents and individual components of a given current suggests further experiments to probe the relationship between membrane structure and channel function in excitable membranes.

Local exchange of oxytocin from the ovarian vein to ovarian arteries in sheep.

Abstract: Local transfer of 125I-labeled oxytocin from the ovarian vein to arteries supplying the ovary, the oviduct, and the tip of the uterine born has been investigated. In five sheep, 10 infusions of 125I-oxytocin over a period of 1 h were performed, and the concentration of labeled polypeptide in the peripheral plasma was compared to ovarian arterial plasma. During 2 consecutive infusions into each animal's ovarian vein, blood was collected simultaneously from the following sites: ovarian branch of the ovarian artery (OBOA), tubal branch of the ovarian artery (TBOA), uterine branch of the ovarian artery (UBOA), and from the jugular vein. In all experiments the concentration of 125I-oxytocin in ovarian arterial plasma was higher than in peripheral plasma. The ratio of ovarian artery/jugular vein for 125I-oxytocin was: OBOA 2.8, TBOA 1.8, UBOA 1.6. Based on a 4 ml/min blood flow through ovarian arteries supplying ovary, oviduct, and the tip of the uterine horn, the local transfer of the total amount of oxytocin infused was estimated to be about 1% (range: 0.1-4.4%). Analysis of variance did not reveal significant differences in the exchange ratios between OBOA, TBOA, and OBOA. However, the variances within these groups are significant, presumably because of anatomical variation in the degree of surface contact area between arteries and veins at the ovarian pedicle. It is concluded that polypeptides are locally recirculated to ovaries, oviduct, and the tip of the uterine horn in a higher concentration than is supplied by peripheral blood. This could provide a mechanism for local distribution and concentration of the ovarian peptides that regulate reproductive function.

Insect olfactory receptor responses to components of pheromone blends

Abstract: Multicomponent pheromone systems are common in many insect species. As our knowledge about the number of different chemical compounds actually involved in a particular communication system increases, so too does the need for an efficient neural mechanism for the encoding of behaviorially relevant odor compounds. Here we consider the electrical activity of olfactory receptor neurons in a subset of the individual pheromone-sensitive sensilla on the antennae of male cabbage looper moths (Trichoplupia ni). Responses to single- and multiple-component stimuli, drawn from seven behaviorally active compounds, were obtained at several different intensities. Some blends elicited electrical responses which were not readily predicted from a knowledge of the receptor neuron's response to individual components.

Differential Structure and Distribution of the High Molecular Weight Brain Microtubule‐Associated Proteins, MAP‐1 and MAP‐2

Abstract: When the first meeting of this kind was held ten years ago, some of the earliest evidence was presented indicating that cytoplasmic microtubules are biochemically complex. It is now well established that these structures consist not only of tubulin, but, in addition, a variety of microtubule-associated proteins, or MAPs. The MAPS were originally identified as a pair of extremely high molecular weight proteins,’ now referred to as MAP-1 and MAP-2’ ( M , 300,000). A considerable number of additional MAPs have since been identified, as discussed elsewhere in this volume. Of all the MAPs, MAP-1 and MAP-2 have received the most attention. They are the most abundant and most readily isolated. In addition, they have proven to be structurally interesting. They appear as fine, fibrous projections on the surface of microtubules,>’ suggesting a role in mediating the interaction of microtubules with other cellular organelles. Electron microscopic images of similar fibers cross-linking microtubules with other organelles in cells have been obtained repeatedly (for example, see references 6 and 7), and a considerable body of physiological and cytological evidence has accumulated indicating that these interactions occur in the living cell. It seems likely that MAP-1 and MAP-2 play a significant role in these interactions. These and the other MAPS are known to have an additional property, that of stimulating microtubule assembly. Some evidence exists that MAPS increase in abundance during neurite outgrowth,8-I0 consistent with a role in the formation or stabilization of new microtubules in the cell.

Progesterone Receptor Replenishment during Sustained Progesterone Treatment in the Hamster Uterus

Abstract: Previous studies have demonstrated that uterine progesterone (P) receptor is under dual hormonal control; estradiol (E2) induces the synthesis of cytosolic P receptor, and P induces the loss of its own receptor and antagonizes E2-induced P receptor replenishment. The objective of this study was to examine the contributions of E2 and P in the replenishment of uterine P receptor during sustained P exposure. Silastic implants of varying length (0.4, 0.8,1.5, 2.5, and 5.0 cm) were packed with crystalline P and placed sc in the flank region of ovariectomized adult female hamsters. The serum P levels obtained with these implants (2–22 ng/ml) were within the physiological range observed previously during the estrous cycle, pregnancy, and lactation in the Syrian hamster. Control animals received a blank implant (1.5 cm). Three, 5, and 7 days after placement of the implants, uterine cytosolic and nuclear P receptor levels were decreased as serum P level was increased by the P implants. Total cellular P receptor le...