Showing papers by "Worcester Foundation for Biomedical Research published in 1989"

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

Abstract: One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.

Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.

Abstract: Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from approximately 90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.

MAP 1A and MAP 1B are structurally related microtubule associated proteins with distinct developmental patterns in the CNS

Abstract: Five high-molecular-weight microtubule-associated proteins (MAPs) were identified in brain tissue in previous work from this laboratory (Bloom et al., 1984). These proteins were termed MAP 1A, 1B, 1C, 2A, and 2B. The MAP 19s differed from the MAP 29s, and showed little evidence of interrelationship on the basis of immunological and biochemical comparison. We report here that MAP 1A and MAP 1B are, in fact, related at the level of subunit composition. Immunoprecipitation of the individual MAPs showed that both contained low-molecular-weight subunits of Mr 30,000 and Mr 19,000 (light chains 1 and 3). An additional subunit, light chain 2 (Mr 28,000), was primarily found in preparations of MAP 1A. The light chains co-sedimented with microtubules after chymotryptic digestion of the MAPs. This suggested an association of the light chains with the microtubule binding domains of the MAPs, which are identified here as distinct fragments of Mr 60,000 for MAP 1A and 120,000 for MAP 1B. A panel of monoclonal anti- MAP 1A and anti-MAP 1B antibodies, including one that reacts with a common phosphorylated epitope, was used to examine the distribution of these proteins in the developing rat brain and spinal cord. MAP 1B was found to be abundant in the newborn brain and to decrease with development, in contrast to MAP 1A which increased with development. By immunohistochemistry MAP 1B was found to be highly concentrated in developing axonal processes in the cerebellar molecular layer, the corticospinal tract, the mossy fibers in the hippocampus, and the olfactory nerve. Of particular interest, the mossy fiber and olfactory nerve staining persisted in the adult, indicating continued outgrowth of the mossy fibers as well as olfactory nerve axons. MAP 1A staining was, in contrast, weak or absent in developing axonal fibers but moderate in mature axons and intense in developing and mature dendritic processes. Our results indicate that MAP 1A and MAP 1B are structurally related components of the neuronal cytoskeleton with complementary patterns of expression.

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues

Abstract: Antisense oligodeoxynucleotides, both the phosphorothioate analogues and unmodified oligomers of the same sequence, inhibit replication and expression of human immunodeficiency virus already growing in tissue cultures of MOLT-3 cells with much greater efficacy than do mismatched ("random") oligomers and homooligomers of the same length and with the same internucleotide modification This preferential inhibitory effect is elicited in as short a time as 4-24 hr postinfection Likewise, antisense oligomers exhibit greater inhibitory effects on human immunodeficiency virus in chronically infected cells than do mismatched oligomers and homooligomers Phosphorothioate antisene oligomers are up to 100 times more potent than unmodified oligomers of the same sequence in these inhibitory assays These results, in major respects, confirm and extend those recently published by Matsukura et al [Matsukura, M, Zon, G, Shinozuka, K, Robert-Guroff, M, Shimada, T, Stein, C A, Mitsuza, H, Wong-Staal, F, Cohen, J S & Broder, S (1989) Proc Natl Acad Sci USA 86, 4244-4248] They also point out the importance of computer analysis of sequences though to be random but that in reality contain significant areas of likely hybridization, either to the viral genome or to the complementary DNA strand synthesized from it They thus reinforce the concept that specific base pairing is a crucial feature of oligonucleotide inhibition of human immunodeficiency virus

Interaction of brain cytoplasmic dynein and MAP2 with a common sequence at the C terminus of tubulin.

Abstract: TWO main types of microtubule-associated proteins (MAPs) have been identified in neuronal cells. The fibrous MAPs, including MAP2 and tau, serve to organize and regulate the assembly of microtubules. A second distinct class of force-producing MAPs, including kinesin1, dynein2–4 and dynamin5, are involved in micro-tubule-based movement. These proteins are mechanochemical ATPases which seem to be responsible for the bidirectional transport of organelles and perhaps also the movement of chromosomes. Here we report that MAP2 inhibits microtubule gliding on dynein-coated coverslips, as well as the microtubule-activated ATPase of dynein, indicating that MAP2 and other fibrous MAPs could be important modulators of microtubule-based motility in vivo. By proteolytic modification of tubulin, we found that dynein interacts with microtubules at the C termini of α- and β-tubulin, the regions previously reported to be the sites for the interaction of MAP26,7. The use of site-directed antibodies implicates a small region of α- and β-tubulin, containing the sequence Glu-Gly-Glu-Glu, as the site of the interaction of dynein and MAP2 with the microtubule.

The basalis of the primate endometrium: a bifunctional germinal compartment.

Abstract: Radioautographic analysis of epithelial and stromal cell proliferation in the primate endometrial functionalis and basalis (rhesus monkey) has identified horizontal zonal patterns of mitotic activation and inhibition during natural menstrual cycles. At 1 h after a single i.v. injection of [3H]thymidine, mitotic activity in endometrial biopsies (hysterotomy) was determined on 9 days from the late proliferative to the late luteal phase (-2 days to + 14 days relative to the estrogen [E2]peak). Labeling indices (LIs) were determined within glandular segments of the 4 horizontal endometrial zones: Transient functionalis Zone I (luminal epithelium) and Zone II (uppermost gland); Germinal basalis: Zone III (middle gland) and Zone IV (basal gland). The size of the dividing epithelial populations (LI) differed zonally. During E2 dominance (-2 days to +3 days), the epithelial LIs of functionalis I (10 +/- 0.3%) and II (9.8 +/- 1.0%) were greater than those of basalis III (5.8 +/- 0.2%) and basalis IV (3.7 +/- 0.8%). During progesterone (P) dominance (+5 days to +14 days), epithelial mitosis was strongly inhibited in functionalis I (4.3 +/- 1.9%), functionalis II (0.8 +/- 0.2%), and basalis III (1.4 +/- 0.5%). Thus germinal basalis III was linked functionally with transient functionalis I and II by periovulatory uniformity in epithelial proliferation and postovulatory mitotic inhibition. A unique mitotic pattern set basalis IV apart from other zones by a steady rise in LI from 1% (-2 days) to 11% (+10 days). The LIs for stromal fibroblasts remained quite uniform in basalis IV but varied in other zones. Thus the postovulatory primate basalis was a distinct bipartite compartment in which the mitotic rate in basalis IV glandular epithelium increased steadily whereas that of basalis III was strongly inhibited. The remarkable enhancement of epithelial mitotic activity in basalis IV may reflect expansion of the stem-progenitor cell population for gestational growth or for post-menstrual regeneration.

Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle.

Abstract: Agrin induces the clustering of acetylcholine receptors (AchRs) and other postsynaptic components on the surface of cultured muscle cells. Molecules closely related if not identical to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a key part in orchestrating synapse regeneration. Agrin or agrin-related molecules are thus likely to play a role in directing the differentiation of the postsynaptic apparatus at the regenerating neuromuscular junction. The present studies are aimed at understanding the role of agrin at developing synapses. We have used anti-agrin monoclonal antibodies combined with alpha-bungarotoxin labeling to establish the localization and time of appearance of agrin-related molecules in muscles of the chick hindlimb. Agrinlike immunoreactivity was observed in premuscle masses from as early as stage 23. AchR clusters were first detected late in stage 25, coincident with the entry of axons into the limb. At this and all subsequent stages examined, greater than 95% of the AchR clusters colocalized with agrin-related molecules. This colocalization was also observed in unpermeabilized whole mount preparations, indicating that the agrin-related molecules were disposed on the external surface of the cells. Agrin-related molecules were also detected in regions of low AchR density on the muscle cell surface. To examine the role of innervation in the expression of agrin-related molecules, aneural limbs were generated by two methods. Examination of these limbs revealed that agrin-related molecules were expressed in the aneural muscle and they colocalized with AchR clusters. Thus, in developing muscle, agrin or a closely related molecule (a) is expressed before AchR clusters are detected; (b) is colocalized with the earliest AchR clusters formed; and (c) can be expressed in muscle and at sites of high AchR density independently of innervation. These results indicate that agrin or a related molecule is likely to play a role in synapse development and suggest that the muscle cell may be at least one source of this molecule.

Preparation of microtubules from rat liver and testis: cytoplasmic dynein is a major microtubule associated protein

Abstract: A microtubule associated protein from brain tissue (MAP 1C), has been found to possess many properties in common with ciliary and flagellar dyneins (Paschal et al.:J. Cell Biol. 105:1273-1282, 1987). However, this protein, now designated as cytoplasmic dynein, exhibited several properties which distinguish it from axonemal forms of the enzyme. We have investigated these characteristics further in a study of cytoplasmic dyneins from non-neuronal tissues. Rat liver and testis in particular were found to contain high levels of cytoplasmic dynein. The yield of dynein from testis was over 70 micrograms/g of tissue, making this the best source of cytoplasmic dynein of all tissues so far examined. The characterization of dynein from these sources has confirmed and extended our previous observations concerning the unique properties of cytoplasmic dynein. Activation of liver and testis dynein occurred at low (less than 1 mg/ml) tubulin concentration. Polypeptides identified as subunits of brain cytoplasmic dynein (74, 59, 57, 55, and 53 kDa) were present in liver and testis preparations. In addition, polypeptides at 150 and 45 kDa were found to copurify with the non-neuronal dyneins. The liver and testis enzyme hydrolyzed pyrimidine nucleotides at rates up to 12.5 times faster than ATP, though the relative affinity of cytoplasmic dynein for CTP was much lower (Km = 1.0 mM) than that for ATP. The properties of the testis enzyme were consistent with its identification as a cytoplasmic dynein rather than a sperm axonemal precursor. These data indicate that cytoplasmic dyneins may be widespread in distribution and that they share certain biochemical properties unique from those of axonemal dyneins. These characteristics are consistent with the proposal that cytoplasmic dynein plays a universal role in retrograde organelle motility.

Regional differences in fat pad responses to short days in Siberian hamsters.

Abstract: Siberian hamsters exhibit decreased body weight and fat after initial exposure to short photoperiods and increased body weight and fat after extended short photoperiod exposure The purpose of the present experiments was to determine if uniform changes in white adipose tissue (WAT) pad weights and lipid metabolism correspond to these short photoperiod-induced changes in body fat Carcass lipid content and testes and fat pad weights [retroperitoneal WAT (RWAT), epididymal WAT (EWAT), and inguinal and dorsal subcutaneous WAT, respectively] were decreased in male hamsters relative to their long day counterparts after 6 and 12 wk of short-day exposure Moreover, EWAT and RWAT weight, EWAT specific lipoprotein lipase activity, and specific and total lipogenesis were disproportionately decreased relative to the subcutaneous fat pads The changes in fat pad weight and metabolism were generally reversed coincident with the return to a long-day-like reproductive status after prolonged short-day exposure (24 and 30 wk) In a less detailed experiment, female Siberian hamsters had decreased body, fat pad, and uterine weights after 6 wk of short-day exposure; however, no fat pad-specific changes in weight were observed The results of these experiments demonstrate that short-day-exposed male Siberian hamsters may be a useful model for examining mechanisms underlying fat pad-specific responses In addition, gender appears to influence the pattern of short-day-induced lipid depletion in this species

Reproductive capacity of sea urchin centrosomes without centrioles

Abstract: For animal cells, the relative roles of the centrioles and the pericentriolar material (the centrosomal microtubule organizing center) in controlling the precise doubling of the centrosome before mitosis have not been well defined. To this end we devised an experimental system that allowed us to characterize the capacity of the centrosomal microtubule organizing center to double regularly in the absence of centrioles. Sea urchin eggs were fertilized, stripped of their fertilization envelopes, and fragmented before syngamy. Those activated egg fragments containing just the female pronucleus assembled a monaster at first mitosis. A serial section ultrastructural analysis of such monasters revealed that the radially arrayed microtubules were organized by a hollow fenestrated sphere of electron-dense material, of the same appearance as pericentriolar material, that was devoid of centrioles. We followed individual fragments with only a female pronucleus through at least three cell cycles and found that the monasters did not double between mitoses. The observation that fragments with only a male pronucleus repeatedly divided in a normal fashion indicates that the assembly and behavior of monasters were not artifacts of egg fragmentation. Our results demonstrate that the activity that controls the precise doubling of the centrosome before mitosis is distinct and experimentally separable from the centrosomal microtubule organizing center. Our observations also extend the correlation between the reproductive capacity of a centrosome and the number of centrioles it contains (G Sluder and CL Rieder, 1985a: J. Cell Biol. 100:887-896). For a cell that normally has centrioles, we show that a centrosome without centrioles does not reproduce between mitoses.

Control of torpor and body weight patterns by a seasonal timer in Siberian hamsters

Abstract: Two experiments were designed to assess whether the short-day-induced patterns of shallow daily torpor, body weight, and other seasonal responses (food intake and pelage pigmentation) exhibited by Siberian hamsters (Phodopus sungorus sungorus) are under the control of a "seasonal timekeeping mechanism" that is independent of reproductive status [testosterone, (T)]. We examined whether the patterning and expression of these seasonal responses were altered by decreases in serum T that accompany gonadal regression during the first 8 wk of short-day exposure (i.e., the "preparatory phase" of the torpor season) or by experimental increases in serum T after this phase. Short-day-housed, castrated hamsters bearing T implants had long-day levels of the hormone and did not exhibit torpor. Appropriate seasonal patterns and levels of torpor, body weight, pelage color stage, and food intake were exhibited after T implant removal although serum T was clamped to long-day levels during the preparatory phase. In animals that were gonad intact during the preparatory phase and were subsequently castrated and given T implants, torpor did not occur as long as the implants were in place. However, the patterns and levels of daily torpor, food intake, and body weight rapidly returned to appropriate seasonal values compared with the castrated, blank-implanted controls on T implant removal; these effects occurred whether the T implants were removed when torpor frequency was increasing, at its peak, or decreasing across the torpor season. T did not affect pelage color stage under any condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Designing, building, and using a fluorescence recovery after photobleaching instrument.

Abstract: Publisher Summary This chapter discusses the design, construction, and proper use of a fluorescence photobleaching recovery (FPR) instrument, the design of spot FPR instruments, and the development of intensified video imaging FPR instruments. The design possibilities of such instruments are essentially infinite. The chapter describes the instruments that are interfaced to personal computers. Such interfacing brings, at reasonable cost to the individual laboratory, the ability to perform both nonlinear least squared data fitting and image processing. Such interfacing requires compromise. However, the rapid evolution of the personal computer in the past 10 years promises continued growth of power and flexibility in the future. FPR is a technique for measuring the lateral diffusibility of macromolecules in membranes and aqueous phases. Many membrane proteins are not completely free to diffuse, and their diffusion rates are too slow to be controlled by lipid fluidity.

Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an upstream TATATA box.

Abstract: Most eukaryotic genes transcribed by RNA polymerase III contain internal control regions. U6 small nuclear RNA genes are transcribed by RNA polymerase III but are unusual in that, at least in vitro, their expression does not require intragenic sequences. Here we show that this is true as well in vivo. A human U6 gene devoid of all but the first 6 and last 10 base-pairs was expressed efficiently after transfection into human 293 cells. We also report data extending the previous identification of 5' flanking sequences important for human U6 gene transcription. Deletion-substitution of a 10 base-pair upstream sequence encompassing the TATATA element (-29 to -24) abolished U6 transcription. A double point mutation in the middle of this element (TATATA-TAGCTA) reduced U6 transcription but not to the extent brought about by TATATA deletion-substitution. These results establish that, in vivo, transcription of human U6 small nuclear RNA is independent of intragenic sequences between nucleotides 6 and 98, and requires the upstream TATATA box.

Paired-pulse facilitation and inhibition in the dentate gyrus is dependent on behavioral state

Abstract: It is well established that neuronal transmission from the entorhinal cortex through the dentate gyrus via the perforant path is dependent on behavioral state. To further study the modulation of neuronal transmission by behavioral state we employed the paired-pulse technique to study interneuronally-mediated inhibition and shortterm facilitation in the dentate gyrus of freely-moving rat preparations. Precisely timed double pulses of electrical stimulation were delivered to the perforant path in the chronically implanted rat preparation during each of four well-defined behavioral states: slow-wave sleep (SWS), REM sleep (REM), immobile waking (IW) or active waking with voluntary movements (AW). Evoked field potentials were recorded in the dentate gyrus and analyzed to measure the population spike amplitude which represents the total number of dentate granule cells firing in synchronous response to perforant path stimulation. The paired-pulse index (PPI) was used as a measure of the net short-term facilitation or interneuronally-mediated inhibition effective at the time of the paired-pulse test and is computed by dividing the amplitude of the second population spike (p2) by the amplitude of the first population spike (p1). During the course of this study 3754 paired-pulse tests were performed in 9 rat preparations. The three interpulse interval (IPI) values used in these studies were 25, 30 and 35 ms. The results showed that the PPI was greater during AW and REM as compared to SWS and IW. The PPI was significantly greater during AW than during SWS and IW regardless of p1 amplitude or IPI value. The PPI was significantly greater during AW than during REM under most conditions except those corresponding to low p1 amplitude and long IPI. The PPIs measured during REM were significantly greater than those measured during SWS and IW at short IPIs (25 and 30 ms) but not at an IPI of 35 ms. These results indicate that short-term facilitation is the dominant response during AW especially when observed using an IPI of 35 ms. In contrast, interneuronally-mediated inhibition was observed to be dominant during SWS and IW. The net effect during REM was observed to lie between these two extremes using an IPI of 25 ms and tended toward short-term facilitation at longer IPIs of 30 and 35 ms. Septal disinhibition of dentate granule cells is proposed as the mechanism for this effect. The behavioral state modulaion of neuronal transmission through the dentate gyrus is discussed in terms of this hypothesis. We conclude that there are probably at least two mechanisms underlying the behavioral modulation of field potentials in the dentate gyrus: (1) an indirect influence modulating the activity of inhibitory interneurons and (2) a more direct influence modulating the excitability of granule cells themselves.

Formation and movement of myosin-containing structures in living fibroblasts.

Abstract: Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, the fluorescent myosin showed a distribution identical to that revealed by immunofluorescence with antimyosin antibodies. Injected myosin became localized in small beads, which were found along large stress fibers, along fine fibers, and in a poorly organized form near the lamellipodia. De novo assembly of beads was observed continuously within or near the lamellipodia, suggesting that myosin molecules may undergo a constant cycling between polymerized and unpolymerized states. The nascent structures then moved away from lamellipodia and became organized into linear arrays. Similar movement was also observed for beads already associated with linear structures, and may represent a continuous flux of myosin structures. The dynamic reorganization of myosin may play an important role in cell movement and polarity.

Culturing cells on the microscope stage.

Abstract: Publisher Summary This chapter discusses the microscope culture system and cell culture chambers. Keeping live, healthy cells on the microscope is crucial for many types of experiments, in particular, fluorescence microscopy of dynamic processes. It is found that although cultured cells may be observed for a short period of time without any special device, a culture system is usually necessary for observations which last for more than 10 min. The major requirements for maintaining the cell are temperature, pH, osmolarity, and nutrients. Cultured mammalian cells should generally be maintained at a temperature close to 37°C. It is observed that with the exception of temperature-sensitive mutants, most cells can tolerate a slightly lower temperature, as well as slight variations in the temperature. The required performance of a microscope culture system depends on the period of observation, the nature of the experiment, and the property of the cell. On the one hand, for short-term observations, the best design may be simply a sealed cover slip. The most sophisticated chambers incorporate mechanisms for the control of various parameters, without the need of a separate microscope incubator. It is found that for open chambers, even with an oil overlay, the pH of the bicarbonate buffer must be maintained by a continuous supply of CO 2 .

Reception of photoperiodic information by fetal siberian hamsters: Role of the mother's pineal gland

Abstract: The rate of reproductive development in juvenile male Siberian hamsters is strongly influenced by daylength (photoperiod). Recent studies indicate that reception of photoperiodic cues begins during fetal life. The present experiments yielded a further demonstration that developing male Siberian hamsters receive information about the photoperiod to which their mother is exposed during pregnancy. The possibility that photoperiodic information is transmitted from mother to young after birth was investigated by cross-fostering young gestated on 12L and 16L to mothers from the other photoperiod. Litters were cross-fostered on the day of birth and then were transferred, along with their foster mothers, to 14L. We found no influence of the mother after birth, indicating that transmission of photoperiodic information from mother to young must occur during gestation. To determine if the pineal gland of the mother is required for this response, adult females were pinealectomized or sham-operated and paired with intact males in 12L, 14L, or 16L. After parturition parents and offspring were exposed to 14L. The influence of prenatal photoperiod on postnatal testicular development in 14L was blocked by pinealectomy of the mother. Postnatal testicular development was retarded in offspring that experienced a photoperiod transfer from either 15L to 14L or 8L to 12L at birth. In contrast, the inhibitory effect of a transfer from 16L to 14L at birth was abolished when juvenile males were exposed to a single long photoperiod (16.3 h light) at age 17-21 days and then were returned to 14L.

Process for synthesizing oligonucleotides and their analogs adaptable to large scale syntheses

Abstract: A process of producing synthetic oligonucleotides on a small or large scale using H-phosphonate nucleoside monomers is described. The process can be used to synthesize oligonucleotides of any length, including oligodeoxyribonucleotides and oligoribonucleotides. The process results in a coupling efficiency of greater than 97% and consumes only two to three equivalents of monomer to activator per coupling reaction. In addition, the process does not require a separate capping step and capping reagent because the activating reagent serves a self-capping function thereby preventing elongation of failed sequences. The H-phosphonate linkages of the fully synthesized oligonucleotide can be oxidized with a variety of reagents to obtain either phosphate diester or other types of modified oligonucleotides.

Effect of melatonin infusion duration and frequency on gonad, lipid, and body mass in pinealectomized male Siberian hamsters.

Abstract: The goal of this study was to discriminate between two hypotheses regarding how the circadian rhythm of pineal melatonin (MEL) production transmits photoperiodic information: (1) A circadian rhythm of sensitivity to MEL regulates the hormone's effect; (2) the duration of the MEL signal, rather than its circadian timing, is the critical parameter of the MEL rhythm. The experiment examined the response of pinealectomized (PINX) male Siberian hamsters to 10-hr (short-day-type) versus 6-hr (long-day-type) duration MEL infusions (10 ng/infusion) in cycles with period lengths (T) of 18, 24, 36, and 48 hr. After cannula implantation, animals were moved from LD 16:8 to LD 10:14 (lights-on from 0500 to 1500 hr, EST), where the timed infusions began. Additional T 24 cycles included as controls employed 18-hr MEL, 18-hr saline (SAL), and 10-hr SAL infusions: Body weight and food intake were measured weekly. After 6 weeks, animals were killed; blood samples were taken for radioimmunoassay (RIA) of serum follicle-stimulating hormone (FSH) and prolactin (PRL); and terminal body, epididymal white adipose tissue (EPIWAT), and paired testis weights were recorded. Six-hour MEL infusions failed to induce short-day-type effects, regardless of the period (T) of the infusion cycle. In contrast, compared to SAL and 6-hr MEL infusions, 10-hr MEL resulted in decreases in body, EPIWAT, and testis weights in T 24, but not in T 36 or T 48. In T 18, testis, body, and EPIWAT mass were decreased, but not to the same extent as in T 24. Similarly, daily 18-hr MEL infusions (T24) were less effective as a short-day stimulus than were 10-hr MEL infusions. The effectiveness of 10-hr, but not 6-hr, MEL infusions in T 18 and T 24 is consistent with the duration hypothesis and argues against the circadian hypothesis. Neither hypothesis could have predicted that all infusion cycles of T greater than or equal to 36 hr, regardless of the infusion durations, would fail to elicit short-day-type responses. This outcome suggests a need for relatively frequent (T less than 36 hr) MEL stimulation in addition to the requirement for adequate duration of each MEL infusion.

Translational status of proenkephalin mRNA in the rat reproductive system.

Abstract: The mRNA for the opioid peptide precursor proenkephalin is widely distributed throughout the male and female reproductive systems of rodents. In the present studies, the concentrations of proenkephalin-derived peptides in selected reproductive tissues of the rat have been determined. When compared with previously characterized tissues such as brain, the peptide contents in reproductive tissues were unexpectedly low relative to the abundance of proenkephalin mRNA. This suggested that either translation of proenkephalin mRNA is relatively inefficient in reproductive tissues or that the turnover of proenkephalin products occurs at a higher rate, or both. To distinguish between these possible mechanisms, the polysomal distributions of proenkephalin mRNA in different rat reproductive tissues and in rat brain were determined. In adult rat testis, in which the predominant proenkephalin RNA is the 1700-nucleotide form present in spermatogenic cells, the transcript was found to be mainly associated with translationally inactive ribonucleoprotein fractions. In contrast, the 1450-nucleotide form of proenkephalin mRNA appeared to be translated to a similar extent in rat brain, epididymis, ovary, and somatic cells of the immature rat testis. It therefore appears that inefficient translation of proenkephalin mRNA in spermatogenic cells is a major determinant of the low ratio of proenkephalin peptides to RNA in the adult rat testis, while posttranslational mechanisms (most likely peptide turnover) are involved in the rat epididymis, ovary, and presumably other reproductive tissues. These findings also indicate that mRNA and/or translation product concentrations within a given tissue are not always accurate indicators of the level of peptide or protein production.

Prostaglandin F2 alpha-stimulated release of ovarian oxytocin in the sheep in vivo: threshold and dose dependency.

Abstract: To determine the threshold of prostaglandin F2 alpha (PGF2 alpha)-stimulated oxytocin secretion from the ovine corpus luteum, low levels of PGF2 alpha (5-100 pg/min) were infused into the ovarian arterial blood supply of sheep with ovarian autotransplants. PGF2 alpha was infused for six sequential 10-min periods at hourly intervals, 6, 12, or 24 days after estrus (n = 3 for each day). Each cycle day was studied during a separate cycle. Oxytocin and progesterone in ovarian venous and carotid arterial plasma was measured by radioimmunoassay, and secretion rates were determined (venous-arterial concentration x plasma flow). In animals treated on Day 6, 5 pg/min PGF2 alpha caused a significant release of oxytocin (p less than 0.01), whereas in animals treated on Day 12, this threshold was 40 pg/min (p less than 0.05). In animals treated on Day 24, the threshold for oxytocin release was greater than 100 pg/min. PGF2 alpha did not significantly change ovarian blood flow or progesterone secretion rate on any day (p greater than 0.05). To determine residual luteal oxytocin after each threshold experiment, 5 mg PGF2 alpha was given i.m. to all animals. Significantly more oxytocin was released by Day 6 than by Day 12 and Day 24 corpora lutea, and by Day 12 than by Day 24 corpora lutea (1.2 micrograms, 0.7 microgram, and 0.3 microgram, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Embryonic expression of a Drosophila src gene: alternate forms of the protein are expressed in segmental stripes and in the nervous system.

Abstract: Expression of the Drosophila src-related gene, Dsrc28C has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Dsrc28C gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Indirect immunofluorescence microscopy reveals that Dsrc28C protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represent the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Dsrc28C cDNA. Staining of embryos with a monoclonal antibody specific for the 66-kD protein indicates that the 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Dsrc28C gene, which exhibit different temporal and spatial patterns of expression in the embryo.