Timing Of Synthesis Research Articles

Calcium and meiotic maturation of the mammalian oocyte

The role of calcium in the regulation of both the meiotic and mitotic cell cycles has been the subject of considerable investigation in the nonmammalian field. In contrast, the mechanisms for signalling meiotic maturation in the mammalian oocyte are not as well documented nor as clearly defined. In the mammalian oocyte, calcium is associated with both spontaneous and hormone-induced meiotic maturation. A transient release of endogenously stored calcium precedes germinal vesicle breakdown and can override cyclic AMP maintained meiotic arrest; it thus may signal the resumption of meiosis. Additionally, extracellular calcium is apparently required for meiotic progression past metaphase I. The time sequence for meiotic resumption and progression is very varied between species. The timing of cell cycle protein synthesis during meiosis suggests that cyclins may be expressed in oocytes of some species much earlier in their development than in others. A generic model is proposed for the mechanism for triggering meiotic resumption in the mammalian oocyte. In this model, the critical components of meiotic resumption involve the temporal relationship of cyclin synthesis and the subsequent activation of the MPF complex by the calcium signal generated, which accounts for differences among species.

Nuclear events during early development in gametophytes of Macrocystis pyrifera, and the temporal effects of a marine contaminant

The time course of DNA synthesis in developing haploid gametophytes of the giant kelp Macrocystis pyrifera was determined, and the effects of arsenic (As) on the temporally distinct nuclear events, DNA synthesis and subsequent nuclear division/translocation, were investigated to establish which of these specific events may be disrupted by this contaminant. Experiments were carried out on material collected from kelp beds near Santa Barbara, California from 1993–1994. Timing of DNA synthesis was determined during development by use of the fluorochrome, DAPI (4′,6-diamidino-2-phenylindole), and single-cell microspectrofluorometry. Zoospores, which result from meiosis, had already undergone two rounds of DNA synthesis at the time of release. The developing gametophytes underwent an additional two rounds of replication of DNA by 16 h of development, and following the first nuclear division/translocation, the gametophyte contained eight times the minimum DNA level throughout subsequent development. Both DNA synthesis and nuclear division/translocation, were found to be inhibited by As. Phosphate enrichment reduced the inhibitory effects of As on division/translocation of the nucleus, supporting the hypothesis that As interferes with phosphorylation. Gametophytes were more severely affected by As under light conditions, as opposed to dark, suggesting that photosynthesis may be more sensitive than dark metabolism.

Differential localization of full-length and processed forms of PF83/AMA-1 an apical membrane antigen of Plasmodium falciparum merozoites

A well conserved 83-kDa apical membrane antigen of Plasmodium falciparum, PF83/AMA-1, is the analogue of PK66/AMA-1, a 66-kDa P. knowlesi protective merozoite protein. PK66/AMA-1 is expressed in late-stage schizonts; is localized within the merozoite apex; and is processed to a 44/42-kDa doublet at, or around, the time of schizont rupture. The processed forms can associate with the merozoite surface. We were interested to further analyze the timing of synthesis and processing, and subcellular localization of PF83/AMA-1, a malaria vaccine candidate, using monoclonal antibodies (mAbs) developed against PF83/AMA-1. Using [ 35S]methionine metabolically labeled asexual blood stage parasites, in combination with indirect single and dual immunofluorescence, we have determined that, in similar fashion to PK66/AMA-1, protein expression of PF83/AMA-1 is restricted to late-stage schizonts with greater than 8 nuclei. PF83/AMA-1 is post-synthetically processed rapidly by cleavage of an N-terminal peptide to a 66-kDa molecule. Both the 83- and the 66-kDa molecules are initially localized at the merozoite apex. In P. falciparum (7G8 strain and CVD-1 clone) the full-length 83-kDa molecule remains apically restricted following merozoite release. However, the processed 66-kDa form can become circumferentially associated with the merozoite surface at or around the time of schizont rupture and merozoite release. After merozoite invasion a processed form of PF83/AMA-1 is present in early ring stage parasites. Comparative analysis of a rhoptry associated protein RAP-l, shows a co-ordinated and compartmentalized release of rhoptry components.

Incompatibility conditioned by the Mla gene in powdery mildew of barley: timing of the effect of cordycepin on hypersensitive cell death

The effect of cordycepin, an inhibitor of mRNA synthesis, on the hypersensitive host cell death response (HR), was tested with epidermal tissues from barley coleoptiles containing the Mla gene for resistance, inoculated with incompatible race 3 of Erysiphe graminis f.sp. hordei . In untreated control tissues, HR occurred 18–30 h after inoculation in 61–91% of cells with haustoria. The percentage of HR was strongly reduced by cordycepin at 10–31 μ m but only if applied 4 h after inoculation or before; not if applied 8 h or later. Effective cordycepin treatments had no inhibitory effect on the development of germ tubes or appressoria and usually increased the development of haustoria and hyphae. The results indicate that mRNAs required for the HR are synthesized in the host within the first 0–8 h after inoculation and that mRNA synthesis is not required thereafter. The timing of protein synthesis required for the HR in the host could not be determined because the inhibitors of protein synthesis used, blasticidin S and cycloheximide, inhibited parasite development at concentrations required to inhibit the HR.

The growth phase-dependent synthesis of cyclopropane fatty acids in Escherichia coli is the result of an RpoS(KatF)-dependent promoter plus enzyme instability.

The formation of cyclopropane fatty acids (CFAs) in Escherichia coli is a post-synthetic modification of the phospholipid bilayer that occurs predominantly as cultures enter the stationary phase of growth. The mechanism of this growth phase-dependent regulation of CFA synthesis was unclear, since log-phase and stationary-phase cultures had been reported to contain similar levels of the enzyme catalysing the reaction (CFA synthase). We report that the timing of CFA synthesis can be explained by two unusual features. Fist, the gene encoding CFA synthase (cfa) was found to be transcribed from two promoters and the 5' ends of both transcripts were mapped by primer extension. One of the promoters was active only during the log-to-stationary phase transition and depended on the putative sigma factor encoded by the rpoS(katF) gene whereas the other promoter had a standard sigma 70 promoter consensus sequence and was expressed throughout the growth curve. Second, CFA synthase activity was shown to be unstable in vivo and a Cfa fusion protein was found to have a half life of < 5 min. The combination of these factors meant that, although CFA synthase was synthesized throughout the growth curve, a large increase in activity occurred during the log-to-stationary phase transition. As stationary phase progressed, the increased CFA synthase activity rapidly declined to the basal level. This transient increase in CFA synthase activity coupled with the cessation of net phospholipid synthesis in stationary phase provides an explanation for the unusual time course of CFA synthesis.

Abrupt RNA changes precede the first cell division during the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro

We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender forms are induced to transform under the same conditions.

Role of Ca2+ in stimulation of DNA synthesis by epidermal growth factor and tumor promoters in cultured rat hepatocytes.

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25-1,800 microM, although the peak of DNA synthesis occurred 5-10 h earlier with 1.2 mM Ca2+ than with 25 microM Ca2+. Complete removal of extracellular Ca2+ using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca(2+)-containing media with Ca(2+)-ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters alpha-hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor-promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]o but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents.

Microspore and pollen development in six male-sterile mutants of Arabidopsis thaliana

Five new recessive male-sterile mutants of Arabidopsis thaliana were isolated following seed mutagenesis by X-rays and ethyl methanesulfonate. The cytology of plants homozygous for the msY and msW mutations suggested that pollen development in these lines became abnormal at or before meiosis. The msK mutation caused faulty timing of synthesis or turnover and distribution of callose. In plants homozygous for the msZ mutation, pollen development failed at a late stage. In wild-type plants, the stamen filament elongated just prior to anther dehiscence. In contrast, in the msZ mutant stamen elongation did not occur. Pollen in msH homozygotes was fertile, but anthers failed to dehisce. The msI mutant of J.H. Van der Ween and P. Wirtz (1968. Euphytica 17: 371 – 377) was included in the present study. Pollen development in this mutant failed shortly after microspore release from tetrads. Complementation tests confirmed that the ms mutations were at different loci. Reduced transmission of certain ms genes was observed. Key words: Arabidopsis thaliana, male sterile mutants, anther dehiscence, callose, inheritance.

Timing of protooncogene expression varies in toxin‐induced liver regeneration

Hepatic expression of the protooncogenes c-fos and c-myc occurs within 2 h after partial hepatectomy, and these immediate early genes are thought to prime the hepatocytes for subsequent proliferation. To examine whether such gene activation occurred in the setting of hepatocyte proliferation after toxic liver injury, protooncogene expression was examined during the regenerative response following liver injury from carbon tetrachloride (CCl4) or galactosamine (GalN). The pattern of protooncogene expression after CCl4 mirrored that seen after partial hepatectomy, with rises in c-fos and c-myc mRNA content within 2 h, and then a rapid return to baseline levels. In contrast, early c-fos and c-myc expression did not occur after GalN injury. Instead GalN-induced regeneration led to a delayed, and prolonged c-fos and c-myc activation which peaked 24-48 h after injury. Increases in c-jun, jun-B, and jun-D mRNA levels also occurred in both models at times similar to the rises of c-fos and c-myc expression. Although the timing of DNA synthesis was identical after GalN or CCl4 treatment, the proliferative response after GalN injury was significantly less than that of CCl4, and marked by the histologic appearance of oval cells. The coadministration of 2-acetylaminofluorene, an inhibitor of differentiated hepatocyte proliferation, together with CCl4 altered the usual pattern of post-CCl4 protooncogene expression to one resembling that seen after GalN injury. Thus, the timing of protooncogene expression during liver regeneration may vary considerably. These variations may influence the nature of the proliferative response in terms of which cell type(s) proliferates, and the amount of regeneration that ensues.

The efficiency and timing of plasmid DNA replication in Xenopus eggs: correlations to the extent of prior chromatin assembly.

Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30-50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg's ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.

Starch synthesis in developing pea embryos.

The pea embryo stores about half of its carbon as starch and has proved to be an excellent system on which to study the nature and regulation of the pathway of starch synthesis. The developing embryo receives its carbon as sucrose, which is metabolized via glycolysis in the cytosol of cotyledonary cells. Glucose 6-phosphate enters the amyloplast - probably via a phosphate-exchange translocator - where it is converted to ADPglucose via phosphoglucomutase and ADPglucose pyrophosphorylase. ADPglucose pyrophosphorylase is the site of action of a mutation at the rb locus, which reduces activity by more than 90 % and the rate of starch synthesis by about 50 %. Study of mutant and wildtype embryos reveals that one of four putative subunits of the enzyme is eliminated by the mutation. Three distinct isoforms of starch synthase catalyze the incorporation of the glucosyl moiety of ADPglucose into starch. Two of these are probably active in the soluble phase of the amyloplast and become incorporated into the granule as it grows, while the third is almost exclusively granule-bound. Analysis of cDNA clones for starch synthases shows that the exclusively granule-bound form is very similar to the 'waxy' gene product believed to be responsible for amylose synthesis in cereal endosperms. The soluble starch synthases show some similarities to the 'waxy' proteins, but clearly belong to a different and previously undescribed class of starch synthases. The pea embryo contains two forms of starch branching enzyme, which are encoded by different genes, are maximally expressed at different times in development, and have different kinetic properties. It is likely that they play different roles in the synthesis of the granule. A mutation at the r locus, which reduces the rate of starch synthesis by about 50% and increases the amylose content of the starch from 30% to 70%, consists of a transposon-like insertion in the gene encoding starch-branching enzyme I. Activity of this isoform is abolished by the mutation. CONTENTS Summary 21 I. Introduction 21 II. The supply of sucrose to the embryo 22 III. The timing and location of starch synthesis 23 IV. The supply of carbon to the amyloplast 23 V. Mutations affecting the committed pathway of starch synthesis 26 VI. The committed pathway 28 Acknowledgements 31 References 31.

Timing of synthesis and cellular localization of two conidiation-specific proteins of Neurospora crassa

The process of conidiation in Neurospora crassa consists of a series of distinct developmental stages culminating in the formation of multinucleate asexual spores called macroconidia. Immunoblotting techniques were used to study the timing of synthesis and cellular localization of CON10 and CON13, the products of two genes that are expressed during conidiation but not during mycelial growth. Both proteins first appear about 8 hr into conidiation; CON10 disappears between 2 and 4 hr after germination. Within conidiating cultures, CON10 and CON13 proteins are localized in conidiophores, with little or no protein present in the underlying mycelium. Immunofluorescence analyses show that CON10 is evenly distributed throughout the cytoplasm of macroconidia. Synthesis of CON10 and CON13 occurs at a time when their specifying mRNAs first appear ( Hager and Yanofsky, Gene 96, 153–159, 1990 ; Sachs and Yanofsky, Dev. Biol. 148, 117–128, 1991 ), suggesting that regulation of synthesis is predominantly transcriptional.

The effect of short-term fasting and a single meal on protein synthesis and oxygen consumption in cod, Gadus morhua

Rates of protein synthesis and oxygen consumption (MO2) in cod were compared in both fasted and refed animals. During a 14-day fast both protein synthesis and respiration rates fell to stable values after 6 days. When a meal of whole sandeel at 6% body weight was fed to fish fasted for 6 days, protein synthesis and MO2 increased to a maximum at between 12 and 18 h after feeding. Peak MO2 was about twice the pre-feeding values, while whole animal protein synthesis increased four-fold. There were differences between tissues in the timing of maximum protein synthesis; the liver and stomach responded faster than the remainder of the body. Maximum protein synthesis rates in the liver and stomach occurred at 6 h after feeding, at which time their calculated contribution to total MO2 was 11%. Similar calculations suggested that the integrated increment in whole animal protein synthesis contributed between 23% and 44% of the post-prandial increase in MO2. It was concluded that protein synthesis is an important contributor to increased MO2 after feeding in cod.

The Timing of Progesterone-Induced Ribonucleic Acid and Protein Synthesis for Augmentation of Luteinizing Hormone Secretion*

Progesterone addition to pituitary cells pretreated with estradiol leads within 45 min to an unambiguous augmentation of pulsatile GnRH-stimulated LH secretion. To investigate this rapid action, we established the kinetics of early events through manipulation of RNA synthesis, protein synthesis, and progesterone-receptor binding. Female rat pituitary cells cultured in medium containing charcoal-treated serum plus 0.2 nM estradiol were changed to 0.1% BSA-medium +/- 200 nM progesterone at time 0; at 90 and 150 min the cells were challenged with 1 nM GnRH 15-min pulses. The 3-fold augmentation of GnRH-stimulated LH secretion induced by progesterone was inhibited completely by simultaneous addition of 1 microM actinomycin D or emetine as was GnRH self-priming. In another series, the ability of cycloheximide to completely block progesterone augmentation was gradually diminished with delay of addition, but even 90 min after progesterone (30 min before GnRH pulse) cycloheximide resulted in 50% blockade of augmentation. In contrast, inhibition of RNA synthesis 60-90 min after progesterone introduction had little or no effect on progesterone augmentation. The temporal profile of inhibition by the progesterone antagonist RU486 was indistinguishable from that resulting from blockade of RNA synthesis and suggests that continual activation of the receptor is required for continued RNA synthesis. In summary: 1) both RNA and protein synthesis are required for GnRH self-priming; and 2) progesterone augmentation of GnRH-stimulated LH secretion requires RNA synthesis and synthesis of protein(s) which appear to be turning over rapidly, accumulating slowly, or both.

Appearance of putative amino acid neurotransmitters during differentiation of neurons in embryonic turtle cerebral cortex

Pyramidal and nonpyramidal neurons can be recognized early in the development of the cerebral cortex in both reptiles and mammals, and the neurotransmitters likely utilized by these cells, glutamate and gamma-aminobutyric acid, or GABA, have been suggested to play critical developmental roles. Information concerning the timing and topography of neurotransmitter synthesis by specific classes of cortical neurons is important for understanding developmental roles of neurotransmitters and for identifying potential zones of neurotransmitter action in the developing brain. We therefore analyzed the appearance of GABA and glutamate in the cerebral cortex of embryonic turtles using polyclonal antisera raised against GABA and glutamate. Neuronal subtypes become immunoreactive for the putative amino acid neurotransmitters GABA and glutamate early in the embryonic development of turtle cerebral cortex, with nonpyramidal cells immunoreactive for GABA and pyramidal cells immunoreactive for glutamate. The results of controls strongly suggest that the immunocytochemical staining in tissue sections by the GABA and glutamate antisera corresponds to fixed endogenous GABA and glutamate. Horizontally oriented cells in the early marginal zone (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble nonpyramidal cells in morphology and distribution. GABA-IR neurons exhibit increasingly diverse morphologies and become distributed in all cortical layers as the cortex matures. Glutamate-immunoreactive (Glu-IR) cells dominate the cellular layer throughout development and are also common in the subcellular layer at early stages, a distribution like that of pyramidal neurons and distinct from that of GABA-IR nonpyramidal cells. The early organization of embryonic turtle cortex in reptiles resembles that of embryonic mammalian cortex, and the immunocytochemical results underline several shared as well as distinguishing features. Early GABA-IR nonpyramidal cells flank the developing cortical plate, composed primarily of pyramidal cells, shown here to be Glu-IR. The earliest GABA-IR cells in turtles likely correspond to Cajal-Retzius cells, a ubiquitous and precocious cell type in vertebrate cortex. Glutamate-IR projection neurons in vertebrates may also be related. The distinctly different topographies of GABA and glutamate containing cells in reptiles and mammals indicate that even if the basic amino acid transmitter-containing cell types are conserved in higher vertebrates, the local interactions mediated by these transmitters may differ. The potential role of GABA and glutamate in nonsynaptic interactions early in cortical development is reinforced by the precocious expression of these neurotransmitters in turtles, well before they are required for synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts.

We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.

Coordination of trophic interactions by separate developmental programs in sensory neurons and their target fields

In the developing vertebrate nervous system the survival of sensory neurons becomes dependent on neurotrophic factors when their axons reach their target fields, and the synthesis of nerve growth factor (NGF) by target field cells commences with the arrival of the earliest axons. The timing of NGF synthesis and the onset of neurotrophic factor dependence are not, however, reliant on innervation. NGF synthesis occurs on time in developing target fields in which innervation is prevented, and sensory neurons cultured before innervating their targets become dependent on neurotrophic factors for survival after a certain length of time in culture. The length of time neurons survive in culture before becoming neurotrophic factor-dependent is related to the time they would normally contact their targets in vivo: populations of neurons that have nearby targets which are innervated early respond to neurotrophic factors before neurons that have more distant targets which are innervated later. The timing of target field innervation is governed not only by the distance axons have to grow but by the rate at which they grow. Axonal growth rate is also regulated in accordance with target distance: neurons with distant targets extend axons faster than neurons with nearby targets. In addition to reviewing evidence for separate developmental programs that control the timing of neurotrophic factor synthesis in the target field and the onset of neurotrophic factor dependence in early sensory neurons, we will consider the mechanisms that might play a role in regulating the survival of neurons during the phase of neurotrophic factor independence.

Nerve Growth Factor Synthesis and Nerve Growth Factor Receptor Expression in Neural Development

Publisher Summary This chapter describes nerve growth factor (NGF) in the context of the neurotrophic theory. It also discusses its implications for understanding the molecular basis of neuron–target interactions in the developing nervous system. Since its discovery, NGF has been the prototype of a class of proteins termed “neurotrophic factors” that regulate the survival and morphology of neurons during development and growth of the vertebrate nervous system. The neurotrophic theory addresses the molecular basis of two aspects of the dependency of neurons on the cells they innervate: the survival of neurons and the growth and arborization of their axons and dendrites. The discovery of exceptionally large amounts of NGF in the submandibular salivary glands of adult male mice has led to substantial progress in understanding the structure and synthesis of this protein. Mouse NGF is a dimer consisting of two identical, 118 amino acid chains each possessing three disulphide bridges. Two major advances have facilitated studies of the site, timing, and regulation of NGF synthesis during development. The first is a very sensitive, specific, two-site enzyme immunoassay for NGF that is capable of detecting the very low levels of this protein present in developing neuronal target fields. The second is the isolation of NGF cDNAs and the subsequent utilization of sensitive hybridization techniques to detect the extremely low levels of NGF mRNA in RNA extracted from tissues and in tissue sections by in situ hybridization.

Distinctive features in the production of IL-6 by human T cells

The production of IL-6 was studied in purified populations of human peripheral blood T cells and monocytes. IL-6 could be elicited from both T cells and thymocytes by a variety of polyclonal stimuli. The expression and production of IL-6 in T cells varied in several ways from that of monocytes: (1) under maximal stimulation, T cells secreted 10-fold less IL-6 than did monocytes; (2) by Western blot, the molecular isoforms produced by T cells were distinct from those produced by monocytes, with at least some isoforms being unique to each cell type; (3) the kinetics of IL-6 expression and production was much slower in T cells than in monocytes; (4) the frequency of labeled cells detected by in situ hybridization was 4% for T cells and 34% for monocytes. Our data indicate that there is considerable diversification among cell types in terms of regulation of IL-6 production and possibly in post-translational modifications. This raises the possibility that the IL-6 produced by different cell types may have varying effects depending on the form produced and the timing of synthesis.