Structure–function studies on hypertrehalosaemic and adipokinetic hormones: activity of naturally occurring analogues and some N- and C-terminal modified analogues

Abstract The relative potencies, with respect to mobilization of lipids in Locusta migratoria L. and in Schistocerca gregaria Forsk. and of carbohydrates in Periplaneta americana L., of eleven naturally-occurring members of the AKH/RPCH-family of peptides and of some analogues that lacked the N-terminal pyroglutamate residue or had a free threonine acid at the C-terminus instead of an amide are assessed. The time courses for carbohydrate and lipid release shown for the peptides suggest that their transport and degradation rates are similar. Therefore, their relative activities reflect most probably the different abilities to bind to the receptors on the fat body cells of the cockroaches and locusts tested. The results show clearly that the N- and C-terminally modified analogues give poor or almost no responses regardless of the bioassay employed. Compared with the structural requirements of the cockroach receptor(s), the receptor(s) of the locusts appear more specific. In the cockroach bioassay, only the two octapeptides Lom-AKH-II and Grb-AKH, both lacking a proline residue at position 6, have high ED₅₀ values, whereas the ED₅₀ values of all other peptides are between 0.4 and 2.7 pmol. The exception is the nonapeptide Mas-AKH, lacking proline and having a serine residue at position 7 (a unique feature compared with the other peptides): no activity is exerted below 100 pmol and only a poor response is elicited with higher dosages. The L. migratoria receptor(s) bind the decapeptides best (exception: Bld-HrTH). Octapeptides, even Scg-AKH-II and Grb-AKH which lack a proline residue, elicit a maximum response, but ED₅₀ values are 7–15-fold higher than with Lom-AKH-I. Lom-AKH-II, however, gives only a truncated response. The nonapeptide Mas-AKH is able to elicit a maximum response, but 50 pmol are needed instead of 2 pmol (Lom-AKH-I). In 5.gregaria a maximum response is achieved with Lom-AKH-I and Grb-AKH, but injections of Scg-AKH-II, Lom-AKH-II and Rom-CC-I result in only truncated responses. The importance of a β-turn in the molecules and the presence of proline is discussed. No evidence is found for an antagonistic effect on binding of those peptides that fail to achieve maximum responses.     

Electroantennogram of the American cockroach: effect of oxygen and an electrical model

1. With a view to clarifying the role of oxygen in the olfactory reception of insects, the intact antenna of the male American cockroach (Periplaneta americana) was studied under normal conditions and during reversible anoxia, external or tracheal, using an electroantennogram (EAG) elicited by a sex pheromone stimulation. As a first step, the anatomical and electrical characteristics of the antenna were investigated. 2. Based on the microanatomical study, a cockroach antenna was characterized by structural parameters reflecting the haemolymph and gas exchange in the antenna. Valves in the wall of a haemolymphatic vessel were discovered. 3. The resting DC background voltage (DCBV) and the EAG were continuously recorded and proved to be more strongly affected by tracheal anoxia than by anoxia from the outside. 4. A minimal electrical model of the antenna based on the Thurm-Kaissling-De Kramer equivalent circuit of a sensillum was shown to be valid to explain the origin of the DCBV and EAG as well as their changes after oxygen cutoff and resumption. 5. Two formal opposing processes, excitation and desensitization, probably related to the receptor mechanisms, have been used to interpret the kinetics of the EAG and the effect of anoxia on EAG parameters. The desensitization is thought to be more sensitive to oxygen lack than the excitation.     

Preliminary studies of Mammea americana L. (Guttiferae) bark/latex extract point to an effective antiulcer effect on gastric ulcer models in mice

Plant extracts are some of the most attractive sources of new drugs and have shown promising results for the treatment of gastric ulcers. Several folk medicinal plants and herbs have been used to treat gastrointestinal disorders, including gastric ulcers. Mammea americana L. (Guttiferae) fruit is very common in the diet of the population of northern South America. Our research interest in this plant arose because of its potential medicinal value as a tonic and against stomachache, as used in folk medicine. In this paper we evaluated three different extracts (ethanolic/EtOH, methanolic/MeOH and dichloromethane/DCM) obtained from M. americana L., for their ability to protect the gastric mucosa against injuries caused by necrotizing agents (0.3 M HCl/60% EtOH), hypothermic restraint stress, nonsteroidal anti-inflammatory drugs (NSAID, indomethacin) and pylorus ligation. In the HCl/EtOH-induced gastric-ulcer model, EtOH and DCM extracts demonstrated significant inhibition of the ulcerative lesion index by 54% (12.0 +/- 2.6 mm) and 86% (3.7 +/- 1.8 mm), respectively, in relation to the control value (26.0 +/- 1.4 mm) (p<0.0001). In the NSAID/cholinomimetic-induced lesion model, both EtOH and DCM extracts showed antiulcerogenic effects with significant reduction in the damage to these gastric lesions of 36% (8.3 +/- 2.0 mm) and 42% (7.5 +/- 1.4 mm), respectively, as compared to the control group (13.0 +/- 0.9 mm) (p<0.0001). In the gastric ulcer induced by hypothermic-restraint stress, both extracts also showed significant activity, and inhibited the gastric lesion index by 58% and 75%, respectively. The EtOH and DCM extracts also changed gastric juice parameters as well as those of cimetidine, decreased gastric acid secretion significantly (p<0.0001), increased pH values and promoted reduced acid output (p<0.0001). In all gastric-ulcer-induced models, MeOH extract did not show any significant antiulcerogenic activity, nor did it change gastric-juice parameters (p>0.05). The results suggest that EtOH and DCM extracts obtained from M. americana possess excellent antisecretory and/or gastrotective effect in all gastric ulcer models. These results suggest that the antiulcerogenic compound(s) present in M. americana may be clustered in the apolar fraction, which will be investigated by our group for the probable mechanisms of action.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Cell recruitment during glial repair: the role of exogenous cells

Summary Selective disruption of the neuroglia in penultimate abdominal connectives of the cockroach nerve is followed by a rapid accumulation of cells in the perineurial layer of the lesion. Subsequently, there is an abrupt, secondary, rise in cell numbers in the undamaged perineurial tissues, anterior to the lesion and adjacent to the 4th abdominal ganglia. By 7 days the increased cell numbers are again effectively confined to the original lesion zone. The initial rise in cell numbers is postulated to result from an invasion by blood-borne haemocytes and the subsequent increase, in undamaged perineurial tissues, from the mobilization of endogenous reactive cells. Recruitment of the endogenous cells is inhibited if the haemocytes are excluded from the lesion. There is a slower mobilization of sub-perineurial cells, which, again, is inhibited following exclusion of haemocytes from the lesion zone. It is postulated that the recruitment of the endogenous reactive cells is initiated by the invading haemocytes which transform to granule-containing cells and release diffusible morphogenic and/or mitogenic factors.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

Role of a novel maintained low-voltage-activated inward current permeable to sodium and calcium in pacemaking of insect neurosecretory neurons

Among ionic currents underlying neuronal pacemaker activity, low-threshold-activated calcium currents contribute to setting the threshold for spike firing. In the insect central nervous system, dorsal unpaired median (DUM) neurons are capable of generating spontaneous electrical activity. It has previously been shown that two distinct (transient and maintained) low-voltage-activated (LVA) calcium currents are responsible for the generation of the pacemaker potential. Whole-cell recordings in voltage- and current-clamp mode were obtained from short-term cultured DUM neurons. Using 100 mM sodium and 2 mM calcium as charge carrier in the external solution as well as conditions that eliminate calcium currents (0.5 mM CdCl₂), voltage-clamp experiments showed that a hitherto unanticipated LVA maintained inward current, activated at around −60 mV, was present. The current amplitude was strongly dependent on internal ATP concentration. Sodium-free solution reduced by 80% the current amplitude. Increasing (5 mM) or decreasing (calcium-free) external calcium concentrations enhanced or reduced, respectively, the maximum conductance without any effect on the voltage dependence. This novel ion channel was permeable to barium but manipulating internal or external magnesium concentrations was without effect on current amplitude or reversal potential. Based on IC₅₀ values, the maintained current was 50-fold less sensitive to TTX than the classical transient voltage-dependent sodium current. Furthermore, it was insensitive to ethosuximide and halothane. Voltage-dependent inactivation analysis revealed an unexpected calcium-sensitive process that involved calcineurin. From these results it appears that, besides the two LVA calcium currents previously described, another LVA maintained inward current permeable to both sodium and calcium was also involved in the generation of the predepolarization. Based on these findings, we propose that a novel calcium-dependent mechanism is involved in the regulation of the pacemaker activity.     

A neuroanatomical study on the organization of the central antennal pathways in insects

Summary Receptor cell axons from the antennal flagellum terminate in the glomeruli of the ipsilateral deutocerebrum in Periplaneta americana and Locusta migratoria. Processes from several groups of deutocerebral neurons also enter the glomeruli and terminate in characteristic branching patterns. There, they contact the antennal axons. Connections are both convergent and divergent. Not only do single central neurons collect the inputs from many receptor cells, but receptor axons were often observed to branch and terminate at more than one deutocerebral neuron. The axons from a portion of the neurons go to form the deutocerebral bundle of the tractus olfactorioglobularis. These axons of the bundle terminate in the ipsilateral calyx of the corpus pedunculatum and in the lateral lobus protocerebri. The processes of the majority of the deutocerebral neurons stay within the deutocerebrum itself and may serve as local interneurons. Part of some antennal fibers terminate in the lobus dorsalis. The lobus glomeratus receives inputs from the maxillary palps and also from processes of deutocerebral neurons.Electron microscopy of synaptic connections and anatomical experiments reveal a complicated pattern of connections between receptor axons and higher order neurons as well as between higher order neurons themselves within the glomeruli.The ratio of the number of antennal fibers to that of relay fibers could easily lead to the interpretation, that the deutocerebrum merely serves as a device for reducing the number of transmission channels. However, coupled with physiological data, anatomical details such as conand divergence of input and interconnections between input channels suggest rather a filtering system and a highly complicated integrative network.     

Structure-activity studies with endogenous allatostatins from Periplaneta americana: expressed receptor compared with functional bioassay

The A-allatostatins (F/YXFGLamides) are insect neuropeptides with inhibitory actions on juvenile hormone (JH) synthesis, muscular contraction and vitellogenesis. They exist in multiple forms within each species. In the cockroach, Periplaneta americana, only one receptor for A-allatostatin has been identified thus far. Here, we have characterised the receptor response to all 15 of the endogenous A-allatostatins encoded by the P. americana allatostatin prohormone gene, together with some analogues, using an indirect heterologous system involving co-expression of the receptor and a potassium channel subunit in Xenopus laevis oocytes and electrophysiological measurements. We have also determined the relative potency of the same peptides to inhibit JH synthesis in corpora allata. Our data reveal that the heterologously expressed receptor responds to all of the endogenous allatostatins and, although differences in potency are recorded, this cannot readily be related to particular differences in the primary structure of the peptides. Similarly, all allatostatins act on the corpora allata to inhibit the synthesis of JH, again with varying potency not readily related to peptide structure. Interestingly, some of the peptides did not perform consistently across the two assays. We show that the receptor is widely expressed in adult P. americana tissues (head, retrocerebral glands, fat body, ovary, male accessory gland, gut, leg muscle, Malpighian tubule and nerve cord) as well as in early larval instars. The spatial expression supports the known pleiotropic activity of allatostatins and role as a paracrine effector. This is the first report of such a detailed characterisation of an invertebrate receptor for allatostatin.     

美洲大蠊内生菌的分离鉴定及酶活性、抑菌作用测定

分离美洲大蠊内生细菌并测定菌体的淀粉、纤维素降解酶活性及抑菌特性。对虫体表面消毒,以LB和高氏一号培养基分离虫体内部细菌并进行16S rRNA基因序列分析。变色圈法测定降解酶活性,抑菌圈法测定抑菌活性。结果共分离到11株细菌,4株属于链霉菌属(Streptomyces),6株分别属于肠杆菌属(Enterbacter)、变形菌属(Proteus）、肠球菌属（Enterococcus）、沙雷氏菌属（Serratia）、芽胞杆菌属（Bacillus）和漫游球菌属（Vagococcus）。另有1株菌在NCBI中与肠杆菌的相似度最高, 为95%,可能代表着一个潜在的新种。4株链霉菌均有淀粉和纤维素降解能力。有3株菌对青枯雷尔氏菌有抑制效果。研究结果表明,美洲大蠊内生菌具有某些功能,甚至含有尚未发现的新菌种,是重要的菌种资源。     

Duplication of a peripheral sensory neuron in the cockroach Periplaneta americana

Summary We have recently examined the electrophysiology and ultrastructure of approximately 100 tactile spines from the metathoracic legs of adult cockroaches. In only one animal the single sensory neuron that innervates the spine was replaced with a pair of apparently identical neurons which we believe were both functional. As far as we are aware this is the first reported study of unprovoked duplication in a peripherally-located insect sensory neuron.Supported by the Canadian Medical Research Council and the Alberta Heritage Foundation for Medical Research     

Postembryonic development of the antennal lobes in Periplaneta americana L.

Summary The postembryonic development of the antennal lobes of Periplaneta americana L. was examined with light- and electron-microscopical methods. There is no difference in the number of glomeruli and neurons in the antennal lobes of larval and adult animals. At hatching, the first larva already possesses the adult number of approximately 125 glomeruli and 500 to 560 deutocerebral neurons in the dorsolateral cell group of each antennal lobe. During postembryonic development the volume of the deutocerebral neurons increases three- to fourfold. The glomeruli of the first larva have about 7 % of the volume of the corresponding adult glomeruli. Since number, pattern, and size ratio of glomeruli (with the exception of the macroglomerulus) are constant in all larval stages and adult animals, it is possible to identify individual glomeruli. During the whole postembryonic development the ordinary glomeruli show a continuous volume increase, which parallels the increase in antennal sensory input. The macroglomerulus develops by way of special growth of two to four neuropil units, but not before the last three to four larval stages and only in males. Its growth precedes the formation of antennal pheromone receptors during the final molt; these receptors are known to project into the macroglomerulus. The development of the macroglomerulus in the last larval stages of the male may be caused by a genetically fixed growth program of specific deutocerebral neurons.Supported by the Deutsche Forschungsgemeinschaft (Scha 291/1)     

Parasitism and decreased response to sex pheromones in malePeriplaneta americana (Dictyoptera: Blattidae)

Parasites are known to alter the behavior of their hosts, but little is known about their effects on responses to sexual stimuli. Periplaneta americanainfected with the acanthocephalan Moniliformis moniliformiswere compared to uninfected animals in their behavioral and electroantennogram responses to a synthetic P. americanapheromone component, periplanone-B, and a pheromone mimic, bornyl acetate. In a t-maze there was no significant difference between infected animals' responses to periplanone-B and a random binomial distribution; the responses of uninfected animals were significantly nonrandom. The electroan-tennogram responses of infected and uninfected animals to bornyl acetate or periplanone-B did not differ significantly, however, indicating that the alteration probably does not occur at the peripheral level but at a central nervous system level.     

New vistas on the initiation and maintenance of insect motor behaviors revealed by specific lesions of the head ganglia

In insects, thoracic pattern generators are modulated by the two head ganglia, the supraesophageal ganglion (brain) and the subesophageal ganglion, which act as higher-order neuronal centers. To explore the contribution of each head ganglion to the initiation and maintenance of specific motor behaviors in cockroaches (Periplaneta americana), we performed specific lesions to remove descending inputs from either the brain or the subesophageal ganglion or both, and quantified the behavioral outcome with a battery of motor tasks. We show that ‘emergency’ behaviors, such as escape, flight, swimming or righting, are initiated at the thoracic level independently of descending inputs from the head ganglia. Yet, the head ganglia play a major role in maintaining these reflexively initiated behaviors. By separately removing each of the two head ganglia, we show that the brain excites flight behavior and inhibits walking-related behaviors, whereas the subesophageal ganglion exerts the opposite effects. Thus, control over specific motor behaviors in cockroaches is anatomically and functionally compartmentalized. We propose a comprehensive model in which the relative permissive versus inhibitory inputs descending from the two head ganglia, combined with thoracic afferent sensory inputs, select a specific thoracic motor pattern while preventing the others.     

Further studies on the antiviral activity of alloferon and its analogues

The subject of our studies was the synthesis, biological evaluation, and conformational studies of insect tridecapeptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) and its analogues such as: [des‐His¹]‐, [Lys¹]‐, [Arg¹]‐, and [Ala¹]‐alloferon. These peptides were synthesized to check the influence of the His residue at position 1 of the alloferon chain on its antiviral activity. Two aspects of the biological effects of these peptides were determined: (i) the cytotoxicity in vitro in the Vero, LLC‐MK2, and HEp‐2 cell lines, and (ii) the antiviral activity in vitro in respect to DNA and RNA viruses. We found that alloferon inhibited the herpes virus multiplication and failed to affect the coxsackie virus replication, whereas [Lys¹]‐alloferon exhibited a high inhibitory action towards both viruses. Moreover, the peptides did not show any cytotoxic activity against the Vero, LLC‐MK2, and HEp‐2 cells. The preliminary circular dichroism conformational studies showed that the peptides investigated seem to prefer an unordered conformation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Embryogenesis of the connective chordotonal organ in the pedicel of the American cockroach: Cell lineage and morphological differentiation

Summary The ontogenesis of single scolopidia of the chordotonal organ of the American cockroach, Periplaneta americana, takes about 4 days. At 23% embryogenesis (100% = 30 d) the first anlagen of scolopidia were identified within the epithelium by staining with anti-horseradish peroxidase. Reconstruction of the cell lineage of the scolopidial cells was facilitated by two facts: (i) the arrangement of the cells throughout ontogenesis follows a strict pattern, and (ii) daughter cells are recognizable for several hours after mitosis by the cytoplasmic bridge and midbody joining them. When they separate, the midbody undergoes lysosomal degeneration in one of these cells. The earliest recognizable stage is a pair of cells, one of which (cell 1) encloses the other (cell 2) apically. The enclosing cell becomes the accessory cell. Cell 2 divides, yielding the mother cell (cell 3) of two sensory cells which degenerate later, and cell 2. Cell 2 gives rise to the attachment cell and to cell 2, which in turn produces the scolopale cell and the mother cell (cell 2 2) of a second pair of sensory cells; the latter are the definitive sensory cells. The end result is the total of 5 cells characteristic of the adult scolopidium. Secretion of the scolopale and cap together with the migration of the sensory cell perikarya into the antennal lumen complete development.     

Parasitoid wasp affects metabolism of cockroach host to favor food preservation for its offspring

Unlike predators, which immediately consume their prey, parasitoid wasps incapacitate their prey to provide a food supply for their offspring. We have examined the effects of the venom of the parasitoid wasp Ampulex compressa on the metabolism of its cockroach prey. This wasp stings into the brain of the cockroach causing hypokinesia. We first established that larval development, from egg laying to pupation, lasts about 8 days. During this period, the metabolism of the stung cockroach slows down, as measured by a decrease in oxygen consumption. Similar decreases in oxygen consumption occurred after pharmacologically induced paralysis or after removing descending input from the head ganglia by severing the neck connectives. However, neither of these two groups of cockroaches survived more than six days, while 90% of stung cockroaches survived at least this long. In addition, cockroaches with severed neck connectives lost significantly more body mass, mainly due to dehydration. Hence, the sting of A. compressa not only renders the cockroach prey helplessly submissive, but also changes its metabolism to sustain more nutrients for the developing larva. This metabolic manipulation is subtler than the complete removal of descending input from the head ganglia, since it leaves some physiological processes, such as water retention, intact.     

Wasp uses venom cocktail to manipulate the behavior of its cockroach prey

The sting of the parasitoid wasp Ampulex compressa is unusual, as it induces a transient paralysis of the front legs followed by grooming behavior and then by a long-term hypokinesia of its cockroach prey. Because the wasp's goal is to provide a living meal for its newborn larva, the behavioral changes in the prey are brought about by manipulating the host behavior in a way beneficial to the wasp and its offspring. To this end, the wasp injects its venom cocktail with two consecutive stings directly into the host's central nervous system. The first sting in the thorax causes a transient front leg paralysis lasting a few minutes. This paralysis is due to the presence of a venom component that induces a postsynaptic block of central cholinergic synaptic transmission. Following the head sting, dopamine identified in the venom appears to induce 30 min of intense grooming. During the long-term hypokinesia that follows the grooming, specific behaviors of the prey are inhibited while others are unaffected. We propose that the venom represses the activity of head ganglia neurons thereby removing the descending excitatory drive to the thoracic neurons.Abbreviations CNS central nervous system - DA dopamine - GI giant interneuron - PSP postsynaptic potential - SEG sub-esophageal ganglion - TI thoracic interneuron     

The mechanosensory apparatus of the femoral tactile spine of the cockroach,Periplaneta americana

Summary Tactile spines are large cuticular sense organs that appear to provide insects with a sense of touch which is spatially coarse but of great sensitivity. Cockroach legs have a number of these spines on each leg and a particularly prominent spine on the end of each femur, the femoral tactile spine. The ease of recording afferent activity from this spine during mechanical stimulation has made it one of the most thoroughly studied insect mechanoreceptors and yet it has never been examined by electron microscopy.We report here the results of an examination of the femoral tactile spine by both scanning and transmission electron microscopy, as well as by light microscopy. The spine is shown to be innervated by a single sensory bipolar neuron with its soma located in the base of the spine. A canal through the wall of the spine leads to the outside and emerges just above the junction between the base of the spine and its articulating socket membrane. The sensory dendrite of the neuron passes from the soma through this canal and forms a modified ciliary sensory ending with the typical dendritic sheath and dense tubular body that is characteristic of insect mechanosensory cuticular sensilla. The tubular body is embedded in a cuticular terminal plug which closes the exterior end of the canal but appears to be fastened to the spine by a very flexible ring of cuticle. This plug is connected to the socket membrane by a specialized socket attachment which presumably serves to move the plug relative to the wall of the spine during movement of the spine within the socket. The morphology of this sensillum is discussed in terms of the possible ways in which it is stimulated by movements of the spine and also in light of the dynamic behaviour of the receptor which is now very well described.Supported by the Canadian Medical Research Council. The authors gratefully acknowledge the expert technical assistance of Sita Prasad and Rodney Gramlich     

Structure-function relationship in hemoproteins: The role of cytochrome c 3 in the reduction of colloidal sulfur by sulfate-reducing bacteria

Cytochromes c ₃ of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c ₃ is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c ₃. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c ₃ is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c ₃ (or another sulfur reductase) than lactate-sulfate-grown cells.