Immunocytochemical localization of opsin, visual arrestin, myosin III, and calmodulin in Limulus lateral eye retinular cells and ventral photoreceptors.

The photoreceptors of the horseshoe crab Limulus polyphemus are classical preparations for studies of the photoresponse and its modulation by circadian clocks. An extensive literature details their physiology and ultrastructure, but relatively little is known about their biochemical organization largely because of a lack of antibodies specific for Limulus photoreceptor proteins. We developed antibodies directed against Limulus opsin, visual arrestin, and myosin III, and we have used them to examine the distributions of these proteins in the Limulus visual system. We also used a commercial antibody to examine the distribution of calmodulin in Limulus photoreceptors. Fixed frozen sections of lateral eye were examined with conventional fluorescence microscopy; ventral photoreceptors were studied with confocal microscopy. Opsin, visual arrestin, myosin III, and calmodulin are all concentrated at the photosensitive rhabdomeral membrane, which is consistent with their participation in the photoresponse. Opsin and visual arrestin, but not myosin III or calmodulin, are also concentrated in extra-rhabdomeral vesicles thought to contain internalized rhabdomeral membrane. In addition, visual arrestin and myosin III were found widely distributed in the cytosol of photoreceptors, suggesting that they have functions in addition to their roles in phototransduction. Our results both clarify and raise new questions about the functions of opsin, visual arrestin, myosin III, and calmodulin in photoreceptors and set the stage for future studies of the impact of light and clock signals on the structure and function of photoreceptors.

Visual arrestin in Limulus is phosphorylated at multiple sites in the light and in the dark.

Arrestins participate in the termination of phototransduction in both vertebrates and invertebrates. However, the visual arrestins of invertebrates and vertebrates differ significantly from one another in that the invertebrate visual arrestins become phosphorylated rapidly in response to light while those in the photoreceptors of vertebrates do not. In an effort to understand the functional relevance of arrestin phosphorylation, we examined this process in the photoreceptors of the horseshoe crab Limulus polyphemus. We report that Limulus visual arrestin can be phosphorylated at three sites near its C-terminus and show that arrestin molecules phosphorylated on one, two, and three sites are normally present in both light- and dark-adapted photoreceptors. Light adaptation increases the amount of arrestin phosphorylated at three sites.

A myosin III from Limulus eyes is a clock-regulated phosphoprotein.

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca2+; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.

Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes.

In rhabdomeral photoreceptors, light stimulates the phosphorylation of arrestin, a protein critical for quenching the photoresponse, by activating a calcium/calmodulin-dependent protein kinase (CaM PK). Here we present biochemical evidence that a CaM PK that phosphorylates arrestin in Limulus eyes is structurally similar to mammalian CaM PK II. In addition, cDNAs encoding proteins homologous to mammalian and Drosophila CaM PK II in the catalytic and regulatory domains were cloned and sequenced from a Limulus lateral eye cDNA library. The Limulus sequences are unique, however, in that they lack most of the association domain. The proteins encoded by these sequences may phosphorylate arrestin.

DNA deaminating ability and genotoxicity of nitric oxide and its progenitors.

Nitric oxide (NO), a multifaceted bioregulatory agent and an environmental pollutant, can also cause genomic alterations. In vitro, NO deaminated deoxynucleosides, deoxynucleotides, and intact DNA at physiological pH. That similar DNA damage can also occur in vivo was tested by treating Salmonella typhimurium strain TA1535 with three NO-releasing compounds, including nitroglycerin. All proved mutagenic. Observed DNA sequence changes were greater than 99% C----T transitions in the hisG46 (CCC) target codon, consistent with a cytosine-deamination mechanism. Because exposure to endogenously and exogenously produced NO is extensive, this mechanism may contribute to the incidence of deamination-related genetic disease and cancer.

Central projections of Limulus photoreceptor cells revealed by a photoreceptor-specific monoclonal antibody.

Studies of lateral, median, and ventral eyes of the chelicerate arthropod Limulus polyphemus (the common American horseshoe crab) are providing important basic information about mechanisms for information processing in the peripheral visual system and for the modulation of visual responses by light and circadian rhythms. The processing of visual information in Limulus brain is less well understood in part because the specific central projections of the various classes of visual neurons are not known. This study describes a mouse monoclonal antibody, 3C6A3, which binds to Limulus photoreceptor cell bodies, their axons, and terminals, but not to any other cell type in the central nervous system. This antibody, and intracellular injection of biocytin, are used to demonstrate the central projections of each type of photoreceptor. Our main conclusions are that: 1) the photoreceptors (retinular cells) of the lateral eye project only to the lamina; 2) the photoreceptors of the lateral rudimentary eye project to both the lamina and medulla; 3) the photoreceptors of the median ocellus project only to the ocellar ganglion; and 4) the photoreceptors of the rudimentary median (endoparietal) eye project to the ocellar ganglion and also into the optic tract. These results, along with previous studies, allow us to infer the projections of the secondary cells. The eccentric cells of the lateral eye project to the lamina, medulla, optic tract, and ocellar ganglion. The arhabdomeral cells of the median ocellus project through the ocellar ganglion and to optic tract to the medulla.

1,3-Dialkyltriazenes: reactive intermediates and DNA alkylation.

The reactions of calf thymus (ct) DNA with 1,3-dimethyltriazene (DMT), N-methyl-N-nitrosourea (MNU), 1,3-diethyltriazene (DET), N-ethyl-N-nitrosourea (ENU), and 1-ethyl-3-methyltriazene (MET) were studied as a function of concentration of the alkylating agents, of various buffers, and of ionic strength. The amount of alkylation at the 7- and O6-positions of guanine increased linearly with dose over a 10-fold concentration range. The slopes of the DMT and MNU curves were identical as were those of DET and ENU. These data suggest that both types of compounds alkylate DNA via a similar intermediate, presumably the corresponding alkanediazonium ion. MET methylates and ethylates DNA, the amount of each product being a function of the competitive formation of the two diazonium ions possible from MET. The MET product ratios could be reproduced by an appropriate mixture of DET and DMT. The alkylation of DNA by DMT and by MET is very sensitive to ionic strength, to the nature of the buffer, and to the identity of the salt used to balance ionic strength. In general, the reaction is favored by low ionic strength, by amine rather than oxy acid buffers, and by doubly charged inert anions. The alkylation of DNA is inversely proportional to the logarithm of the ionic strength over a wide range. The mutagenic activity of triazenes in Salmonella typhimurium is correlated very well with the ability of the triazenes to form adducts, particularly O6-guanine adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine: a putative afferent neurotransmitter in Limulus eyes.

Histamine has been proposed as a photoreceptor neurotransmitter in two major groups of arthropods, the insects and the crustacea. In this study biochemical and immunocytochemical approaches were used to examine the synthesis, endogenous content, and cellular distribution of histamine in the visual system of the horseshoe crab Limulus polyphemus, an ancient chelicerate arthropod. Studies with this animal have been critical to our understanding of the basic processes of vision. High-voltage paper electrophoresis was used to assay for histamine synthesis in Limulus tissues incubated with radiolabeled histidine; histamine synthesis was detected in the lateral, median, and ventral eyes and optic nerves and in the visual centers in the brain. Endogenous histamine, assayed as its orthophthalaldehyde derivative by high-performance liquid chromatography and electrochemical detection, was also detected in these tissues. Immunocytochemical analyses, with an antiserum directed against a protein conjugate of histamine, revealed histamine-like immunoreactivity in the somata of photoreceptors in each of the eyes and in the regions of the brain where the photoreceptors terminate. Histamine-like immunoreactivity was also intense in the cell bodies and axon collaterals of eccentric cells in the lateral eye and in eccentric cell projections in the brain. These results show that histamine is a major biogenic amine in the Limulus visual system, and they suggest that this amine is involved in transmitting visual information from the eyes to the brain and in lateral inhibition, a fundamental mechanism for processing visual information in the lateral eye.

Aerial oxidation of hydrazines to nitrosamines.

When 1,1-dimethylhydrazine and N-aminopiperidine were deliberately exposed to air substantial amounts of the corresponding carcinogenic nitrosamines were formed. Unoxidized samples of 1,1-dimethylhydrazine were not mutagenic while oxidized samples (which contained much higher levels of nitrosamines) were mutagenic. Both unoxidized and oxidized samples of N-aminopiperidine were mutagenic.

Efferent Innervation to Limulus Eyes In Vivo Phosphorylates a 122 kD Protein.

Efferent fibers innervate all of the eyes of the horseshoe crab, Limulus polyphemus. Driven by a circadian clock located in the central nervous system, the activity of the fibers at night is responsible for anatomical, biochemical, and physiological changes in the eyes, which increase their ability to detect and respond to light. We showed previously that octopamine, a putative efferent neurotransmitter, stimulates the phosphorylation of a 122 kD protein in in vitro preparations of both ventral and lateral eyes by means of a cAMP-dependent mechanism. We now report that phosphorylation of the 122 kD protein in the lateral eye is enhanced in vivo: (1) at night, in correlation with efferent nerve input activated by the circadian clock; and (2) during the day, in response to electrical stimulation of efferent axons. We show further that the 122 kD protein is enriched in, and may be restricted to, tissues that contain photoreceptors. We postulate that this protein is involved in the efferent-stimulated increase in retinal sensitivity.

Degradation and disposal of some antineoplastic drugs.

Bulk quantities and pharmaceutical preparations of the antineoplastic drugs carmustine (BCNU), lomustine (CCNU), chlorozotocin, N-[2-chloroethyl]-N'-[2,6-dioxo-3-piperidinyl]-N-nitrosourea (PCNU), methyl CCNU, mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, uracil mustard, and spiromustine may be degraded using nickel-aluminum alloy in KOH solution. The drugs are completely destroyed and only nonmutagenic reaction mixtures are produced. Destruction of cyclophosphamide in tablets requires refluxing in HCl before the nickel-aluminum alloy reduction. Streptozotocin, chlorambucil, and mechlorethamine may be degraded using an excess of saturated sodium bicarbonate solution. The nitrosourea drugs BCNU, CCNU, chlorozotocin, PCNU, methyl CCNU, and streptozotocin were also degraded using hydrogen bromide in glacial acetic acid. The drugs were completely destroyed but some of the reaction mixtures were mutagenic and the products were found to be, in some instances, the corresponding mutagenic, denitrosated compounds.

Case-control study of colorectal cancer and fecal mutagenicity.

Fecal mutagenicity was measured in 68 patients with colorectal cancer and in 114 controls, using Salmonella tester strains TA98 and TA100 with and without S9 activation. Samples were also tested for fecapentaenes by high-performance liquid chromatography, to permit the separation of fecapentaene and non-fecapentaene mutagenicity. Overall, no significant case-control differences in fecal mutagenicity were observed. However, when samples containing high concentrations of fecapentaenes were excluded, non-fecapentaene TA98 mutagenicity was observed in eight cases (12%) and only four controls (4%), resulting in an estimated relative risk of 4.4 (95% confidence interval = 1.0-21.1). The association of colorectal cancer risk with non-fecapentaene TA98 mutagenicity could not be explained as an artifact of diagnostic workup or gastrointestinal bleeding among the cases. Smoking could also be excluded as a source of the TA98 mutagenicity seen, but possible dietary origins are still being explored.

Decomposition and quality control considerations in biological work with fecapentaene preparations.

Solutions of synthetic fecapentaene 12 (FP-12) intended for carcinogenicity studies were found to decompose extremely rapidly during customary dosage procedures. Apparent half-lives as short as 15 min were observed. While rates and even the qualitative course of decomposition were surprisingly variable in replicate experiments, high concentration and exposure to air were confirmed to be especially important destabilizing influences. The results suggested a primary role for a radical decomposition mechanism in the presence of atmospheric oxygen. Consistent with this hypothesis, FP-12 solutions were significantly stabilized by the radical chain-breaking antioxidant vitamin E. On the other hand, dithiothreitol greatly destabilized FP-12, presumably because of its nucleophilicity. The diacetyl diester of FP-12 was more soluble than the parent diol, but its decomposition rates in the presence and absence of vitamin E were similar to those of unesterified FP-12. Ultraviolet irradiation of an all-trans-FP-12 solution decreased its concentration by 70% in 0.5 min. The mutagenicities of the decomposition/isomerization products of FP-12, as studied in Salmonella typhimurium tester strain TA 100, ranged from negligible to comparable with all-trans-FP-12 itself. It is concluded that unchecked decomposition of fecapentaene preparations can profoundly affect biological tests therewith. While this can be largely controlled through the use of rigorous precautions, including protection from air, light, nucleophiles, and acids as well as selection of the lowest concentration compatible with the application at hand, the data argue strongly for inclusion of appropriate quality control measures in all future dosing operations to prove that the biological activity reported is that of the fecapentaene itself rather than that of a decomposed dosing solution.

Fecapentaene concentration and mutagenicity in 718 North American stool samples.

The fecapentaenes (FP) are the predominant fecal mutagens identified to date, but they have not been shown to be carcinogenic. Epidemiologists looking for other fecal mutagens that may be related to colorectal cancer must disentangle from their investigations the pervasive mutagenic effect of the fecapentaenes. As a first step to studying the epidemiology of fecal mutagenicity independent of fecapentaenes, we compared FP measurements and Salmonella mutagenicity assay results for 718 acetone-extracted stool samples collected from a variety of subjects in the Washington DC metropolitan areas. In this large group, 50% of mutagenic samples contained elevated fecapentaenes. Specifically, three-quarters of the samples mutagenic in TA100 contained high FP levels. In contrast, mutagenicity in TA98 was not generally explainable by fecapentaenes, suggesting that non-fecapentaene TA98 mutagenicity should be one focus of future efforts to uncover colorectal carcinogens of etiologic importance.

Decontamination and disposal of nitrosoureas and related N-nitroso compounds.

An improved procedure for chemically decontaminating residues of nitrosoureas and related N-nitroso compounds ("nitrosamides") commonly used in the cancer research laboratory is proposed. Treatment of accumulated wastes with aluminum:nickel alloy powder while progressively increasing the basicity of the medium consistently led to at least 99.98% destruction of each nitrosamide tested. Hazardous diazoalkanes were never detected in yields of greater than 0.1%. The mutagenicity of the completed reaction mixtures was never more than 3 times background except when the N-nitroso compound contained a 2-chloroethyl group. In most cases, the completeness of reaction could be determined chromatographically, not only to demonstrate the disappearance of the starting N-nitroso compound, but also to follow production of identifiable products in sufficient abundance to account for the starting material destroyed; none of the organic products observed was mutagenic in any of the four tester strains used. The procedure described herein proved reliable in two checker laboratories besides our own when applied to mixtures of seven N-nitroso compounds: N-methyl-N-nitroso-p-toluene-sulfonamide; N-methyl-N-nitrosourethane; N-methyl-N-nitrosourea; N-methyl-N'-nitro-N-nitrosoguanidine; N-ethyl-N-nitrosourea; N-ethyl-N'-nitro-N-nitrosoguanidine; and N-ethyl-N-nitrosourethane. All of the other procedures investigated for destruction of nitrosamides, including the widely used approach of dissolving the nitrosamides in alkali, were associated with important disadvantages.

Biochemical epidemiology of cervical neoplasia: measuring cigarette smoke constituents in the cervix.

In preparation for an epidemiological investigation of cigarette smoking and cervical neoplasia, we studied methods of measuring cervical exposure to tobacco smoke. The measurement of cotinine in cervical flushes by radioimmunoassay proved to be highly accurate in distinguishing smokers from nonsmokers, achieving 100% sensitivity and 97% specificity. In most subjects, quantitative levels of cervical cotinine and nicotine mirrored recent smoking intensity. Some of the apparent exceptions may have resulted from metabolic/secretory traits of the subjects. If so, the biochemical measurement of smoke constituents in the cervix might prove more valuable for epidemiological studies of cervical neoplasia than data on current smoking behavior collected by interview.

Relative mutagenic and prophage-inducing effects of mono- and di-alkyl nitrosoureas.

Mutagenic capacity, prophage-inducing ability, and decomposition rates of mono- and di-alkylnitrosoureas with the same alkyl groups were studied in aqueous systems using S. typhimurium strain TA1535 and E. coli strain BR339 (lambda). Slower decomposition rates of nitrosodialkylureas compared with monoalkylnitrosoureas were not reflected in reduced mutagenicity, with the exception of the methylating agents. With the monoalkylnitrosoureas, mutagenicity varied with length of the alkyl chain, and was greatly increased by oxygen substituents. Among the dialkylnitrosoureas, mutagenesis and decomposition rates were dependent on the substitutent in the N-1 position, adjacent to the nitroso group. Prophage induction, on the other hand, was dependent on a substituent in the N-3 position. The results suggested the existence of two distinct mechanisms for DNA damage, one SOS-dependent and the other SOS-independent, by which different dialkylnitrosoureas acted. Neither in vitro assay system was a reasonable model for the carcinogenic activity of these compounds.

Quantitative structure-activity relationship of the mutagenicity of substituted N-nitroso-N-benzylmethylamines: possible implications for carcinogenicity.

The relative mutagenicities of substituted N-nitroso-N-benzylmethylamines have been reexamined from a quantitative structure-activity relationship point of view. Most of the compounds were mutagenic toward Salmonella typhimurium TA 1535 with Aroclor-induced male hamster liver S9 activation. The dose-response data were subjected to a multiple linear regression equation calculated in a stepwise manner, which found that the differences in mutagenicities could be explained primarily by differences in the three-bond path molecular connectivity index, with smaller contributions from sigma and pi. Moreover, a polynomial regression analysis showed that the maximum mutagenicity could be explained by an optimal amount of electron withdrawal by the substituent which would cause a weakening, or activation, of the methylene C-H bond. The possible relevance of these observations to carcinogenesis is discussed.