Silencing synaptic communication between random interneurons during Drosophila larval locomotion.

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons. These larvae were screened for aberrant behavior at an elevated temperature (31-32°C). Among larvae with abnormal locomotion or sensory-motor responses, some had very small numbers of GFP-labeled temperature-sensitive interneurons. Labeled ascending interneurons projecting from the abdominal ganglia to specific brain neuropile compartments emerged as candidates for mediation of larval locomotion. Random targeting of small sets of neurons for functional evaluation, together with anatomical mapping of their processes, provides a tool for identifying the regions of the central nervous system that are required for normal locomotion. We discuss the limitations and advantages of this approach to discovery of interneurons that regulate motor behavior.

Drosophila Amphiphysin is a post-synaptic protein required for normal locomotion but not endocytosis.

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.

Melatonin and melanocyte functions.

The effect of melatonin on melanocyte functions was studied by incubating whole-skin organ cultures with melatonin, as well as by assessing melatonin positivity in melanocytes versus dendricity and pigmentation, when arrested in the G(2) phase. From this study, it was observed that melatonin positivity is inversely related to the length of UV exposure. Increasing melatonin levels are related to decreasing dendricity and pigment donation during photoresponse in the G(2) phase. Melanocyte melatonin positivity increases with dark incubation and is higher with a pulse of UV exposure after dark incubation with melatonin. This increase is associated with a doubling of melanocyte number after dark incubation and a further doubling upon exposure to a pulse of UV. The melanocytes directly take up melatonin, which results in a marked increase in their numbers. Thus, extreme caution should be exercised when using melatonin as an anticancer drug. This finding also simulates the melanocyte repopulation of the skin with repigmentation during summer in polar animals.

Behavioral characterization and genetic analysis of the Drosophila melanogaster larval response to light as revealed by a novel individual assay.

A new assay was designed, named checker, that measures the individual response to light in the fruitfly Drosophila melanogaster larva. In this assay the Drosophila larva apparently modulates its pattern of locomotion when faced with a choice between a dark and lit environment by orienting its movement towards the dark environment. We show that, in this assay, a response to light can be measured as an increase in residence time in the dark versus the lit quadrant. Mutations that disrupt phototransduction in the adult Drosophila abolish the larval response to light, demonstrating that this larval visual function is similar to that of the adult fly. Similarly, no response to light was detected in strains where the larval visual system (photoreceptors and target area) was disrupted by a mutation in the homeobox containing gene sine oculis (so) gene. Ablation of photoreceptors by the targeted expression of the cell death gene hid under the control of the photoreceptor-specific transcription factor glass (gl) abolishes this response entirely. Finally, we demonstrate that this response to light can be mediated by rhodopsins other than the blue absorbing Rh1.

The tamas gene, identified as a mutation that disrupts larval behavior in Drosophila melanogaster, codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-gamma125).

From a screen of pupal lethal lines of Drosophila melanogaster we identified a mutant strain that displayed a reproducible reduction in the larval response to light. Moreover, this mutant strain showed defects in the development of the adult visual system and failure to undergo behavioral changes characteristic of the wandering stage. The foraging third instar larvae remained in the food substrate for a prolonged period and died at or just before pupariation. Using a new assay for individual larval photobehavior we determined that the lack of response to light in these mutants was due to a primary deficit in locomotion. The mutation responsible for these phenotypes was mapped to the lethal complementation group l(2)34Dc, which we renamed tamas (translated from Sanskrit as "dark inertia"). Sequencing of mutant alleles demonstrated that tamas codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-gamma125).

Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions.

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. We propose the existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors. Our results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene.

Novel antitumor 2-cyanoaziridine-1-carboxamides.

A set of 20 2-cyanoaziridine-1-carboxamides was synthesized from 2-cyanoaziridine and appropriate isocyanates. These compounds were active against a variety of solid and hematological tumor cells in culture, including strains resistant to doxorubicin and mitoxantrone. Their potencies in these assays correlated with the lipophilicity of substituents. The N-phenyl derivative was more potent and equally effective to imexon, a cyclized 2-cyanoaziridine-1-carboxamide of clinical interest, against cloned fresh human tumors.

A simultaneous ex vivo model of embryogenesis: I. Organogenesis.

In vivo culture of chick embryo was carried out to develop an experimental interphase between in vitro and in vivo study of embryonic physiology. In the process, a simultaneous model of vasculogenesis and organogenesis has been worked out, which is impossible to achieve in mammalian system. Both early (40 hours of incubation) and late (64 & 88 hours of incubation) hours of cultures were conducted for morphological and morphometric studies. A new combination of stains was used in place of conventional haematoxylin and eosin in 40 hours old whole-mount of embryos. Semithin plastic sections were etched for haematoxylin/pyronin stain in addition to paraffin (both normal and enblock) sections. Specific stains (histological, enzyme histochemical or immunohistochemical) were chosen according to the specific organs/areas studied. Immunohistochemistry and NADPH-diaphorase activity were standardized in whole-mount of embryos. Morphometry was done using camera lucida and quantitative image analysis system. A parallel preparation of extra embryonic whole-mounts, paraffin and semithin plastic sections with different types of stainings provides evidence for the scope of the simultaneous study of vasculogenesis. Thus the morphological and morphometric data presented in this and the succeeding article describe the scope and avenues for the use of ex vivo model in various aspects of embryonic physiology, preliminary drug trials/metabolism, radiology, teratology and toxicology.

A simultaneous ex vivo model of embryogenesis: II. Vasculogenesis.

Vasculogenesis was simultaneously studied with embryogenesis in in ovo chick embryo culture, which was harvested at 40 hours. Endodermal cells and vascular endothelial cells were studied using a new combination of stains, immunohistochemistry (for nuclei and basement membrane) and NADPH-diaphorase activity in whole-mounts, paraffin sections and etched semithin sections. The model can be used for the study of developmental process of blood vessels as well as embryonic physiology of blood vessels vis-a-vis organogenesis in response to different angiogenic agents, drug trials, cancer therapy by angiostatic chemicals/radiations and toxins. Considering that vasculogenesis/angiogenesis as one of the fundamental phenomena in physiology, pathophysiology, toxicology and pharmacology of developmental sciences, the model in developing embryo is presented.

The hair follicle: a specialised UV receptor in the human skin?

Coat colour changes in polar animals are related to seasonal variation in photic inputs. The present work was performed to study the photoresponses of hair follicular melanocytes in human skin. The melanocytes, being photosensitive cells, can function as UV biosensors, since dendrites extend towards the source of UV light. Fifty-one skin biopsies from the margin of vitiligo were subjected to whole skin organ cultures. These were exposed to a pulse of UV light to study hair bulb melanocytes in vitiligo. It is observed that the melanocytes are seen within the anagen matrix. These melanocytes are poorly dendritic in control and dark-incubated cultures. On UV exposure, they become highly dendritic, the dendrites extending towards the hair shaft in 93.5%. They show prominent catechol oxidase and noradrenaline positivity, all features of UV responsiveness. The melanocytes within the hair follicle are not directly exposed to UV light. The melanocyte dendricity and the alignment of dendrites towards the shaft on UV exposure indicate that the columns of the cells in the hair shaft act as an efficient fibre-optic system, transmitting UV light. Morphologically, the keratinocytes in the hair shaft are arranged in compressed linear columns which resemble the coaxial bundles of commercial fibre-optic strands as is observed in plants. Keratinocytes in the inner and outer sheaths do not show this arrangement. Thus the hair follicle functions as a specialised UV receptor in the skin responding to nuances of photic inputs in human skin. This is reflected in coat colour changes in animals exposed to large variations in day-night cycles.

The role of melanocytes in the repair of UV related DNA damage in keratinocytes.

Epidermal pigmentation and UV exposure are related to the incidence of skin tumors. There is a higher incidence of UV related skin tumors in populations with low pigment and in vitiligo patients, resulting from DNA damage. Normally DNA repair processes set in with the expression of PCNA in the keratinocyte. The present study was conducted on the marginal zone skin in vitiligo. Whole skin organ cultures irradiated with increasing doses of UV in the 280400 nm range show that in the depigmented area there is no expression of PCNA by the keratinocytes. In comparison, the marginal zone keratinocytes show a dose related positivity in the presence of UV responsive melanocytes. These photoresponsive melanocytes show dendricity and cytoplasmic PCNA positivity. The melanocytes interact with keratinocytes by active melanosome transfer. From this study it is suggested that this involves transfer of PCNA as well. The present study indicates the differentiating keratinocytes in skin do not express PCNA but appear to be dependent on active UV responding melanocytes for DNA repair. This factor could play an important role in the occurrence of UV-related skin tumors.

The UV-responsive melanocyte system: a peripheral network for photoperiodic time measurements. a function of indoleamine expression.

Earlier studies indicate the involvement of indoleamines in the melanocyte photoresponse and cell cycle. In this study whole skin organ cultures were done to study the location of indoleamine expression during the photoresponse. Whole skin organ cultures from marginal zone vitiligo were incubated in MEM containing adriamycin and exposed to varying pulses of UV at 2 h of incubation. The G2 phase marginal melanocytes show increasing dendricity in response to increasing UV exposure at 3 h of incubation. On immunohistochemical staining for serotonin and melatonin, it is observed that both are positive in these melanocytes. The proportion of serotonin-positive melanocytes rises with increasing UV exposure while that of melatonin positivity rises with decreasing UV exposure, thus simulating the pineal response to light entrainment. This is due to photoinhibition of enzymes converting serotonin to melatonin. This study shows that the melanocytes in the skin can serve as the peripheral neural net for photoperiodic time measurements - the biological calendar.

Photomodulation of the melanocyte cell cycle by indoleamines.

Melanocytes are photoresponsive cells which respond to varying doses of UV exposure in the G2 phase of the cell cycle by prominent dendricity. This photoresponse is related to indoleamine light sensitivity. The present study highlights the role of indoleamines in the photomodulation of the melanocyte cell cycle. The study was conducted on 40 whole-skin organ cultures taken from the marginal zone of vitiligo. Twenty organ cultures were subjected to G2-phase arrest, while 20 were incubated in tryptamine. The organ cultures were incubated in the dark, exposed to a pulse of 120 s UV at 2 h of incubation and harvested 3 and 6 h after UV exposure. It has been reported that the photosensitive enzymes N-acetyl transferase (NAT) and hydroxyindole-o-methyl transferase (HIOMT) are activated during the G2 phase. The conversion of serotonin to melatonin is inhibited by UV exposure as seen at 3 h. This activity recovers on continued dark incubation 6 h after UV exposure. On incubation with tryptamine, UV exposure results in utilisation of tryptamine as seen by prominent indoleamine positivity. Three hours after UV exposure, there is 75% dendricity indicating G2 phase traverse. There is a corresponding high serotonin positivity with a low melatonin positivity. This is reversed following 6 h of dark incubation with high melatonin positivity indicating reactivation of NAT and HIOMT. This is accompanied by a doubling of the melanocyte number due to mitotic traverse and an arrest in G1 phase with low utilisation of tryptamine. Thus tryptamine is utilised by melanocytes on UV exposure to be synchronised and traversed into G2 phase activating the photosensitive enzymes NAT and HIOMT. When followed by a dark phase, melatonin accumulates to traverse the melanocytes through M-phase of the cell cycle with doubling of the cell number. Thus the uptake and metabolisation of indoleamine precursors photomodulate the melanocyte cell cycle on UV exposure.

1,4-disubstituted anthracene antitumor agents.

Three different types of 1,4-disubstituted anthracenes were synthesized, and their cytotoxicity in a panel of tumor cells was compared with that of the corresponding 9,10-disubstituted anthracenes. The panel contained human myeloma, melanoma, colon, and lung cancer cells and sensitive and multidrug-resistant murine L1210 leukemia cells. These compounds had [[(dimethylamino)ethyl]amino]methyl, N-[(dimethylamino)ethyl]carbamoyl, and carboxaldehyde (4,5-dihydro-1H-imidazol-2-yl)hydrazone side chains. The 1,4-diamide was more potent across the tumor panel than the corresponding 9,10-isomer, but the 1,4-diamine and the 1,4-hydrazone were less potent than their 9,10-isomers. Although the 1,4-hydrazone was active against P388 leukemia in mice, it was inactive against L1210 leukemia. Within each pair of compounds, the one with greater average potency against tumor cells gave a greater increase in the transition melt temperature of DNA.

UV-dependent melanocyte plasticity--the structure-function relationship.

The UV response of marginal melanocytes in vitiliginous skin was studied using a whole skin organ culture technique. This method assesses the plasticity of melanocytes in response to UV. It is observed that there is a sequential increase in catecholoxidase production and in the volume and dendricity of the melanocytes on exposure to a single pulse of UV, reaching a peak at 3 1/2 h. From this study it is evident that the melanocyte shows a prominent structural and functional plasticity in response to UV. Implicit is the utilisation and transduction of UV energy by the melanocyte, for transcriptional and translational activity, enhancing catecholoxidase and cell structural protein production.

6- and 7-substituted 2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h] isoquinoline-1,3-diones: synthesis, nucleophilic displacements, antitumor activity, and quantitative structure-activity relationships.

New 2-[2'-(dimethylamino)ethyl]-3H-dibenz[de,h]isoquinoline-1,3-diones with substituents at the 6- and 7-positions were prepared. Nucleophilic aromatic displacement was a key reaction in the syntheses. Ten of the new compounds were more potent than the unsubstituted compound, azonafide, in a panel of tumor cells including human melanoma and ovarian cancer and murine sensitive and MDR L1210 leukemia. They also were less cardiotoxic in cell culture. Four of these compounds were not cross-resistant with the MDR leukemia, and one of them, 6-ethoxyazonafide, was nearly as potent against solid tumor cells as leukemia cells. These compounds also had good potency against human breast, colon, and lung cancer cells, including doxorubicin and mitoxantrone resistant cell lines. Advantages of the new analogues over azonafide were less in vivo, but 6-ethoxyazonafide was more effective against L1210 leukemia and subcutaneous B16 melanoma in mice. Although correlations of antitumor potency in cells and physicochemical properties of substituents were not found, there were statistically significant correlations of DNA melt transition temperature (delta Tm) with potency in solid tumor cells and sensitive and MDR resistant L1210 leukemia cells for 6-substituted azonafides and with solid tumors for 7-substituted azonafides.

ACTH acts directly on melanocytes to stimulate melanogenesis--an in vitro study.

A total of 108 whole skin organ cultures taken from vitiliginous skin were incubated in MEM containing ACTH. It was observed that 53.7% that is 58 showed a positive response with an increase in pigment production and enzyme activity, as observed on frozen sections stained for dopaoxidase activity. On immunohistochemical staining for locating ACTH binding, it is observed that 27.3% control skin and 72.7% ACTH treated skin show positivity. The ACTH is seen to bind with the melanocyte membrane as well as the cytoplasm. This indicates that ACTH can bind to the MSH-receptors expressed by the melanocyte. Thus, ACTH acts directly on the melanocyte to enhance melanogenesis and does not require to act via the adrenal-pituitary axis. This also indicates that the response is not associated with immune suppression by ACTH.

Corticotropin expression by human melanocytes in the skin.

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G2-phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV-sensitivity of the melanocytes.

Aspirin by virtue of its acidic property may act as teratogen in early chick embryo.

Aspirin (acetylsalicyclic acid) was dissolved either in normal saline or in phosphate buffer and was used in two doses to find out whether teratogenic potential of aspirin in chick blastoderm model is due to its acidic property or due to drug action. Drug was injected sub-blastodermally by window technique in fresh embryonated eggs after 17 hours of incubation at 39 degrees C. Eggs were re-incubated and harvested at 40 hours. Normal development of embryos was seen with normal saline and percentage of normal embryos with 30 micrograms (pH-3.19) and 120 micrograms (pH-2.64) aspirin was 31.7 and 4.9 respectively. Buffer produced 80.8% normal embryos and buffered 30 micrograms (pH-6.87) and 120 micrograms (pH-6.69) aspirin produced 67.7% and 30.8% normal embryos respectively. Changing the pH of aspirin to near neutral decreased the defect induced by aspirin but a significant effect of aspirin was observed at higher dose which could be independent of pH action.

Expression of proliferating cell nuclear antigen (PCNA): proliferative phase functions and malignant transformation of melanocytes.

The UV-dependent G2-phase functions of melanocytes include dendricity, the expression of melanocyte stimulating hormone (MSH) receptors and neural differentiation. The present report studied highly dendritic melanocytes in epidermis overlying tumours, seborrhoeic keratosis, basal cell carcinoma and melanomas. The expression of the proliferative protein PCNA was studied by immunohistochemistry, as this indicates cells in S/G2-phase. In the non-neoplastic dendritic melanocytes, PCNA is retained in the cytoplasm, resulting in the arrest of the cells in the S/G2-phase for prolonged periods, as indicated by the length and complexity of the dendritic processes. In melanomas, this barrier is overcome with rapid proliferation of the cells and loss of dendricity. PCNA is produced in the cytoplasm and transported into the nucleus during the S-phase, as observed in melanomas. The arrest of melanocytes in the S/G2-phase for long periods associated with UV responsiveness makes these cells vulnerable to DNA damage and neoplasia. Pools of PCNA in the cytoplasm, when transported into the nucleus, would support the rapid proliferation observed in melanomas.