Early formation of sexually dimorphic glomeruli in the developing olfactory lobe of the brain of the moth Manduca sexta

The antennal lobes (ALs), the primary olfactory centers, of the moth Manduca sexta are sexually dimorphic. Only ALs of males possess the macroglomerular complex (MGC), the site of primary processing of information about the female's sex pheromone. To understand the development of identified, odor-specific olfactory glomeruli, we investigated the cellular events involved in the morphogenesis of the MGC by means of various fluorescence staining techniques and laser-scanning confocal microscopy. The MGC lies near the entrance of the antennal nerve into the AL of the adult male and comprises three glomeruli, the globular cumulus and two toroidal structures. The MGC forms during early stages of metamorphic adult development through a stereotyped sequence of coordinated changes in MGC-specific receptor axons, glial cells, and early-ingrowing projection neurons of the medial group of AL neurons. The MGC divisions are the earliest glomeruli to form in the male AL, and their basic organization is established within about 3 days after ingrowth of the first sensory axons. Despite their special anatomical features, the MGC glomeruli develop in a manner similar to that of the ordinary glomeruli. Comparison of the ALs of males and females reveals that two relatively large and early-developing glomeruli that are situated dorsolaterally in the female AL appear to be female-specific. Development of the sexually dimorphic glomeruli diverges immediately after the ingrowth of the first olfactory receptor axons, resulting in the formation of these large glomeruli in females and the MGC in males.     

Development of adult thoracic leg muscles during metamorphosis of the hawk moth Manduca sexta

During metamorphosis, the larval thoracic legs of the hawk moth Manduca sexta are replaced by a new set of adult legs. The larval leg motoneurons persist to innervate new adult muscles, and the motor terminals remain within the developing adult legs. Here we describe the fate of the larval leg muscles and the origin of new muscles within the adult legs. During the larval instars, large and small nuclei proliferate within leg muscle fibers. Near the end of the larval stage a subset of the small nuclei undergo a wave of proliferation, as indicated by the incorporation of 5-bromodeoxyuridine, whereas other nuclei die. However, none of the larval leg muscles fibers persist to serve as templates for adult muscle formation, and there was no evidence for persistence of larval myonuclei. Migrating myoblasts that are born within aggregate to form adult muscle anlagen at specific production sites within the developing imaginal legs. Intense nuclear proliferation occurs within the anlagen during the early pupal stage, followed by muscle fiber formation and striation. We conclude that adult leg muscles form mainly, if not exclusively, from migrating myoblasts that without the involvement of larval elements.     

Innervation regulates the metamorphic fates of larval abdominal muscles in the moth, Manduca sexta

With the onset of metamorphosis, the abdominal muscles of the moth, Manduca sexta, follow one of three developmental fates: maintenance, respecification, or death. The maintained muscles retain their larval size and morphology throughout adult development. The respecified and dying muscles dedifferentiate, which involves regression, nuclear degeneration, and myofibril breakdown. Nuclei in both dying and respecified muscles also proliferate. The amount of nuclear degeneration is greater in the dying muscle fibers, and the amount of nuclear proliferation is greater in the respecified muscles. Four to ten days after pupation, the sizes of the respecified muscles stabilize while the dying muscles are lost. During regression, a subset of the respecified muscle fibers die. The surviving respecified muscle fibers grow and differentiate during the last half of adult development. In respecified muscles, denervation triggers an increased amount of nuclear degeneration and a decreased amount of nuclear proliferation. As a result, denervated respecified fibers experience increased muscle regression including an increased loss of muscle fibers and sometimes muscle death. Surviving respecified fibers still grow and differentiate yet are only 5 to 12% of the control size. Denervation triggers dedifferentiation in maintained muscles, resulting in fiber loss and occasionally muscle death. The percentage of fibers which dedifferentiate varies between different muscles. Denervation also triggers nuclear proliferation, with the amount of nuclear proliferation correlated with the extent of dedifferentiation of the individual muscle fibers. The dedifferentiated maintained fibers subsequently undergo differentiation in the absence of muscle growth.     

Development and organization of a nitric-oxide-sensitive peripheral neural plexus in larvae of the moth, Manduca sexta

Each hemisegment of the Manduca sexta larva is supplied with a subepidermal plexus of approximately 350 multidendritic neurons. An initial set of neurons, the primary plexus neurons, arise at 35-45% of embryogenesis. These neurons comprise 12-16 uniquely identifiable neurons per hemisegment that have homologues in other insect larvae. Each spreads processes across a characteristic portion of the body wall and has an axon that projects into the central nervous system. Secondary plexus neurons are born in two waves: the first between 70% and 80% of embryogenesis and the second during the molt to the second larval stage. The secondary plexus neurons are multidendritic, spread uniformly across the body wall, and appear to make contacts with the primary plexus neurons. Each secondary plexus cell arises as part of a five-cell cluster; the other cells produce a sensory bristle and socket along with the bristle sensory neuron and a glial cell. Application of nitric oxide (NO) donors induces plexus neurons to produce cyclic 3',5' guanosine monophosphate (cGMP), suggesting the presence of soluble guanylate cyclase. With few exceptions, plexus neurons become sensitive to NO stimulation approximately 10 hours after their birth and remain so throughout larval life. Cyclic GMP is detected primarily in the cytoplasm of plexus neurons and extends into the finest peripheral dendrites. Our results suggest that cGMP participates in the development and/or physiology of this peripheral neural plexus.     

Ultrastructure of the neurosecretory cells in the brain of diapausing pupae of the tobacco hornworm, Manduca sexta (L)

The ultrastructure of seven different types of neurosecretory cells (NSC) found in the medial and lateral areas of the brain of diapausing Manduca sexta is described. The five different types of NSC in the medial area have characteristic differences in their shape, size, neurosecretory granules (NSG), and the morphology of their organelles. The cell types of the medial area accumulated the NSG, but did not appear to be synthesizing and packaging new NSG, whereas the NSC in the lateral region were synthesizing and packaging NSG during diapause. The possible significance of the relationship between the lateral and medial cells is discussed.     

Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF-alpha on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF-alpha in the presence of interferon-gamma (IFN-gamma) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN-gamma, no increase in Mn-SOD activity was detected. The induction of IFN-gamma-dependent Mn-SOD activity by anti-Fas antibody or TNF-alpha was concentration-dependent; the maximal effect was observed at 1-10 micrograms/ml and 5-10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF-alpha in the presence of IFN-gamma resulted in an additive increase in Mn-SOD activity. Although the addition of 12-o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN-gamma-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF-alpha. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN-gamma and that TPA augments the process through the activation of protein kinase C.     

Insulin receptor-like tyrosine kinase in the tobacco hornworm, Manduca sexta

Urinary bacterial mutagenicity was used as a biomarker of exposure to ambient air pollution in a group of women working outdoors in the city of Teplice (TP; Northern Bohemia) with higher levels of air pollution than a similar group of women in the city of Prachatice (PT; Southern Bohemia). The Salmonella typhimurium plate incorporation assay with the TA98 and YG1041 strains and microsuspension assay with the YG1041 strain were used for testing the urinary mutagenicity. PAH and their metabolites were analyzed by HPLC and GC/MS methods. The significantly higher values of most PAHs/metabolites detected in a TP group confirmed the differences of PAH exposures between both groups. In the plate incorporation assay, the TA98 strain was not able to detect the increase in urinary mutagenicity, but, for the YG1041 strain, the urinary mutagenicity was clearly determined with a significant difference in number of YG1041 + S9 revertants between the TP and PT groups. The microsuspension assay increased the mean response by about 10-fold over the standard plate test; however, no statistical difference between TP and PT groups was found due to high interindividual variability and small sample size. Comparing the urinary PAH/metabolites to urinary mutagenicity, significant correlations were observed between the plate incorporation mutagenicity results with the YG1041 revertants in the presence of metabolic activation and several of the urinary PAH/metabolites. On the contrary, in the microsuspension assay, several urinary PAH/metabolites correlated significantly with the YG1041 revertants only in the absence of metabolic activation. This may indicate the influence of different treatment conditions of assays on the urinary mutagenicity results. The results suggest the insufficient sensitivity of the TA98 tester strain to determinate low urinary level of mutagens. On the contrary, the use of the YG1041 tester strain increases the probability of detecting an effect of environmental exposure and seems to be applicable to biological monitoring. To definitely replace the standard plate incorporation assay with the microsuspension method is not possible without further comparative studies.     

Thermal sensitivity of growth and feeding in Manduca sexta caterpillars

We explore how the thermal sensitivity of organismic performance emerges from the thermal sensitivity of the underlying component processes involved, using growth and feeding of Manduca sexta caterpillars as a model system. We measured thermal performance curves for the short-term rates of growth, consumption, protein (casein) digestion, amino acid (methionine) uptake, and respiration in fifth-instar caterpillars over a biologically realistic temperature range from 14 degrees to 42 degrees C. Growth and consumption rates increased between 14 degrees and 26 degrees C, reached a maximum value near 34 degrees C, and declined rapidly above 38 degrees C. In contrast, protein digestion rate and respiration rate increased monotonically over the entire temperature range, and amino acid uptake rate increased with temperatures up to 38 degrees C and then leveled off between 38 degrees and 42 degrees C. These results suggest that the shape and position of the thermal performance curve for growth rate--in particular the maximum at 34 degrees C and rapid decline above 38 degrees C--was most closely correlated with the thermal sensitivity of consumption rate; the declining growth performance above 38 degrees C was not associated with declines in digestion or uptake rates or with accelerated respiration rates at these temperatures.     

Postembryonic neurogenesis in the central nervous system of the tobacco hornworm, Manduca sexta. III. Spatial and temporal patterns of proliferation

Postembryonic neurogenesis leads to a dramatic increase in the number of functional neurons within the segmental ganglia of the moth, Manduca sexta. These adult-specific neurons are generated during larval life by segment-specific arrays of individually identifiable stem cells, or neuroblasts (Nbs). By the end of the feeding larval stage, each Nb has generated a discrete nest of progeny, which ranges in size from less than 10 to more than 70 progeny. The sizes of these identifiable nests of progeny vary in a segment-specific manner, with the thoracic nests containing a greater number of progeny compared with their homologues in the simpler abdominal ganglia. In order to describe those factors that influence the size of the post-embryonic neuronal lineages, we examined the spatial and temporal pattern of postembryonic neurogenesis in the segmental ganglia of Manduca. The rates at which the identifiable nests accumulated progeny were estimated by counting the number of progeny within the nests, using sectioned material isolated from animals at stages ranging from embryonic hatching until the end of the feeding larval stage. All of the postembryonic Nbs began to generate progeny at around the time of the molt to the third larval instar. Each nest added progeny at a rate that was a characteristic of its identity and segment of origin. Although all of the nests within the thorax continued to accumulate progeny throughout the feeding larval stage, several of the abdominal nests showed little or no growth following the molt to the fifth larval instar. The thymidine analog 5-bromo 2-deoxyuridine (5-BrdU) was used to estimate the mitotic rates of the identifiable Nbs. The number of labeled progeny within a nest 24 h after application of 5-BrdU ranged from a low of 1 to 2 to a high of 11 to 13 labeled cells. In some instances there was a good correlation between the estimated mitotic rate of an identified Nb and the rate of growth of its associated nest of progeny. However, several of the identifiable nests accumulated progeny at a slower rate than predicted based on the estimated mitotic rate of the Nb. Cell death appears to be responsible for slowing the growth of the nests during the feeding larval stage. We estimate that 10% to 70% of the neurons generated during the feeding larval stage degenerate within 24 h of their birth. The level of cell death observed within a nest was dependent on both its identity and its segment of origin.     

Absence of short-term regulation over gluconeogenesis by glucose in the insect Manduca sexta L

In vivo gluconeogenesis from (3-13 C)alanine was evident in terminal instar Manduca sexta larvae from the selective fractional 13C enrichment in trehalose, a disaccharide of glucose and the major blood sugar of insects. De novo glucose synthesis was observed in insects fed a low carbohydrate diet for 1 or more days. Gluconeogenesis was not inhibited by a single injection of glucose nor by short-term feeding on glucose-supplemented diet. Reduced fractional 13C enrichment in trehalose was demonstrated upon glucose administration, but was explained by isotopic dilution following direct synthesis of trehalose from the unlabeled glucose. Isotopic dilution was also quantified by analysis of the 13C labeling pattern in trehalose synthesized following injection of (1,2-13C2)glucose. The results suggest the absence of short-term regulation over gluconeogenesis by glucose and may partially explain why blood sugar level in M. sexta and other insects fluctuates over a wide concentration range. Although glucose had no observable effects on gluconeogenesis, injection of or feeding glucose resulted in a significantly increased activity of the pentose phosphate pathway.     

Oxidized collagen stimulates proliferation of vascular smooth muscle cells

We have examined the use of presurgical morphine-midazolam combination in 80 children aged 2-10 y undergoing repair of hypospadias. They were allocated randomly, in a double-blind study, to receive one of four morphine-midazolam combination doses (n = 20 each); (group I: 75 microg/kg each) [corrected] (group II: 75 microg/kg [corrected] morphine, 50 microg/kg [corrected] midazolam); (group III: 50 microg/kg [corrected] morphine, 75 microg/kg [corrected] midazolam); (group IV: 50 microg/kg [corrected] each). Drugs were given after induction of anesthesia and before the start of surgery. Observational scoring system, using crying, movement, agitation, posture and localization of pain as scoring criteria, was used to assess the children during their stay in the recovery room together with their sedative and/or analgesic requirement. Pre-surgical morphine-midazolam administration produced stable hemodynamic variables with satisfactory postoperative analgesia suggesting 75 microg/kg [corrected] dose of both morphine and midazolam as upper permissible dose, and 50 microg/kg [corrected] each as lower effective dose.     

Programmed cell death in the tobacco hornworm, Manduca sexta: alteration in protein synthesis

The metamorphic death of the labial glands of the tobacco hornworm, Manduca sexta, occurs during a 4 day period during larva-to-pupa metamorphosis. The earliest changes marking the death of the cell, all occurring on the first day, are a sharp drop in protein synthesis, coupled with the selective survival or upregulation of a few messages. An early rearrangement of the rough endoplasmic reticulum is presumably related to the generalized decrease in protein synthesis. Lysosomal acid phosphatase also begins to increase very early, and ultimately the bulk of the cytoplasm is destroyed in autophagic vacuoles, but activation of lysosomes does not account for the decreased rate of synthesis. The mechanism by which most protein synthesis is depressed remains under investigation.     

Leptin stimulates the proliferation of murine myelocytic and primitive hematopoietic progenitor cells

Leptin, the product of obese gene, was originally identified as a factor regulating body-weight homeostasis and energy balance. The present study has shown that leptin acts on murine hematopoiesis in vitro. In the culture of bone marrow cells (BMC) of normal mice, leptin induced only granulocyte-macrophage (GM) colony formation in a dose-dependent manner, and no other types of colonies were detected even in the presence of erythropoietin (Epo). Leptin also induced GM colony formation from BMC of db/db mutant mice whose leptin receptors were incomplete, but the responsiveness was significantly reduced. The effect of leptin on GM colony formation from BMC of normal mice was also observed in serum-free culture, and comparable with that of GM-colony-stimulating factor (CSF ). Although leptin alone supported few colonies from BMC of 5-fluorouracil (5-FU)-treated mice in serum-free culture, remarkable synergism between leptin and stem cell factor (SCF ) was obtained in the colony formation. The addition of leptin to SCF enhanced the SCF-dependent GM colony formation and induced the generation of a number of multilineage colonies in the presence of Epo. When lineage (Lin)-Sca-1(+) cells sorted from BMC of 5-FU-treated mice were incubated in serum-free culture, leptin synergized with SCF in the formation of blast cell colonies, which efficiently produced secondary colonies including a large proportion of multilineage colonies in the replating experiment. In serum-free cultures of clone-sorted Lin-c-Kit+Sca-1(+) and Lin-c-Kit+Sca-1(-) cells, although synergism of leptin and SCF was observed in the colony formation from both cells, leptin alone induced the colony formation from Lin-c-Kit+Sca-1(-), but not Lin-c-Kit+Sca-1(+) cells. These results have shown that leptin stimulates the proliferation of murine myelocytic progenitor cells and synergizes with SCF in the proliferation of primitive hematopoietic progenitors in vitro.     

Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the Moth, Manduca sexta

Molecular dynamics simulation in explicit solvent and continuum solvent models are applied to investigate the relative stability of A- and B-form helices for two DNA sequences, dA10-dT10 and dG10-dC10 in three structural forms. One structural form is based on an unrestrained molecular dynamics (MD) trajectory starting from a canonical B-DNA structure, the second is based on a MD trajectory starting in a canonical B-DNA structure with the sugars constrained to be C2'-endo and the third simulation started from a canonical A-DNA structure with the sugars constrained to C3'-endo puckers. For the energetic analysis, structures were taken as snapshots from nanosecond length molecular dynamics simulations computed in a consistent fashion in explicit solvent, applying the particle mesh Ewald method and the Cornell et al. force field. The electrostatic contributions to solvation free energies are computed using both a finite-difference Poisson-Boltzmann model and a pairwise Generalized Born model. The non-electrostatic contributions to the solvation free energies are estimated with a solvent accessible surface area dependent term. To estimate the gas phase component of the relative free energy between the various structures, the mean solute internal energies (determined with the Cornell et al. molecular mechanics potential including all pairwise interactions within the solute) and estimates of the solute entropy (using a harmonic approximation) were used. Consistent with experiment, the polyG-polyC (GC) structures are found to be much more A-phillic than the polyA-polyT (AT) structures, the latter being quite A-phobic. The dominant energy components responsible for this difference comes from the internal and van der Waal energies. A perhaps less appreciated difference between the GC and AT rich sequences is suggested by the calculated salt dependence which demonstrates a significantly enhanced ability to drive GC rich sequences towards an A-form structure compared to AT rich sequences. In addition to being A-phobic, the AT structure also has a noticably larger helical repeat than GC and other mixed sequence duplexes, consistent with experiment. Analysis of the average solvent density from the trajectories shows hydration patterns in qualitative agreement with experiment and previous theoretical treatments.     

Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro

Nicotine is a major component of cigarette smoke and has been postulated to play an important role in atherogenesis and malignancy. Endothelial cell growth may be regulated by nicotine, yet operative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine (10(-14)-10(-4) M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in the presence and absence of hydroxyurea (an inhibitor of scheduled DNA synthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF and PDECGF, respectively). Nicotine significantly stimulated endothelial cell DNA synthesis and proliferation at concentrations lower than those obtained in blood after smoking (<10(-8) M). The stimulatory effects of nicotine were enhanced by serum (0.5%) and PDECGF and were blocked by the nicotinic-receptor antagonist hexamethonium. The response to nicotine was bimodal because cytotoxicity was observed at higher concentrations (>10(-6) M). This study has implications for understanding cellular mechanisms of nicotine action. The results may be important in tumor angiogenesis, atherogenesis, and vascular dysfunction in smokers.     

Follicular fluid of women with endometriosis stimulates the proliferation of endometrial stromal cells

The peritoneal environment in endometriosis is known to have growth-promoting effects on endometrial cells. To investigate whether follicular fluid, a contributor to the peritoneal fluid, stimulates endometrial cell proliferation, we incubated endometrial stromal cells in culture with various dilutions of follicular fluid obtained from women with or without endometriosis undergoing oocyte retrieval for in-vitro fertilization. Cell proliferation assays were performed using follicular fluid from 28 women (without endometriosis, n = 13; with endometriosis, n = 15) in eight different endometrial stromal cell culture set-ups. Cell proliferation was assessed by a colorimetric method. Maximum cell proliferation was detected when endometrial cells were incubated with 50% dilution of follicular fluid for 48 h. Follicular fluid from women with endometriosis induced significantly higher cell proliferation than follicular fluid from women without endometriosis (P < 0.05). Our findings indicate that follicular fluid contents may contribute to the growth-promoting factors in the peritoneal fluid of women with endometriosis.     

Functional activation of glial cells in early and delayed episodes of the brain damage

The authors have developed a rapid and convenient method for purification of a low molecular weight form (delta 10) of the bacterial plasminogen activator, staphylokinase. Recombinant staphylokinase is expressed in Escherichia coli, with an amino terminal extension that facilitated purification by immobilized metal-affinity chromatography. Purified staphylokinase is treated with human plasminogen, and the resulting truncated form is purified using a combination of immobilized metal affinity chromatography and hydrophobic interaction chromatography. Purified protein is characterized by amino terminal sequencing and in vitro plasminogen activation assay.