Postembryonic proliferation in the spiny lobster antennular epithelium: rate of genesis of olfactory receptor neurons is dependent on molt stage

Olfactory systems undergo continuous growth and turnover in many animals. Many decapod crustaceans, such as lobsters and crayfish, have indeterminate growth, and in these animals, turnover of both peripheral and central components of the olfactory system occurs continuously throughout life. In this study, we examine the dynamics of olfactory receptor neuron (ORN) proliferation in the antennule of the Caribbean spiny lobster, Panulirus argus, using in vivo incorporation of the cell proliferation marker BrdU. We show that addition of ORNs occurs in a "proximal proliferation zone" (PPZ), which exists on the proximo-lateral margin of the existing ORN population. The PPZ is spatially and temporally dynamic in that it travels as a wave in the proximal and lateral directions in the antennule. This wave results in continuous addition of ORNs throughout the molt cycle. The rate of proliferation, as measured by the size and shape of the PPZ, changes depending on the animal's molt stage. The rate is highest during premolt and lowest during intermolt. ORNs are the most prominent cell-type produced in the PPZ, but other cell types, including glia, are also produced. Patches of proliferating epithelial cells occur immediately proximal to the PPZ, suggesting that neuronal and glial precursors reside in this region. Possible mechanisms for peripheral and central modulation of ORN development are discussed.     

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.     

Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy

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Modulation of behavior by biogenic amines and peptides in the blue crab,Callinectes sapidus

Using the blue crab Callinectes sapidus as a model system, we have investigated the effects of potential neuromodulators on freely behaving animals. Of interest is the modulatory effect of a number of drugs on three rhythmic behaviors of the blue crab: courtship display (CD) of the male crab, sideways swimming and backward swimming. The drugs tested were proctolin, dopamine, octopamine, serotonin, and norepinephrine. Injection of each drug elicited a unique posture or combination of limb movements. These experiments showed two results pertinent to CD behavior: A posture identical to the CD posture was displayed after dopamine injection; and rhythmic leg waving similar to CD was evoked by proctolin. An unusual combination of flexion and extension of all limbs and movements of some limbs occurred after serotonin injection. Injection of octopamine led to a posture antagonistic to CD posture. The effects of these drugs were concentration- and time-dependent. Injection of dopamine, octopamine, or serotonin produced effects that were seasonally-dependent, and the influence of proctolin proved to be dependent on developmental stage. Quantitative analysis of leg waving movements after proctolin injection allowed for comparison of these movements to naturally-occurring behavior.Abbreviations CD courtship display - DA dopamine - OA octopamine - 5-HT serotonin - NE norepinephrine - PROC proctolin     

Behavioral olfactory discrimination of mixtures in the spiny lobster (Panulirus argus) based on a habituation paradigm

A habituation paradigm was used to examine behavioral aspectsof discrimination of stimulus quality in olfaction in the spinylobster (Panulirus argus). Magnitude of search response to twoconcentrations of artificial mixtures of crab, oyster, mulletand shrimp and to artificial seawater was measured in five lobstersbefore and immediately after habituation to crab mixture. Habituationto crab mixture was accomplished through 2-min presentationsof 5 ml of alternating concentrations (0.05 and 0.5 mM) of crabmixture stimulus, repeated every 5 min for a total durationof at least 3 h. This procedure resulted in at least a 42% decreasein response to any mixture, but the decrease was greatest forcrab mixture, the habituating stimulus. A habituation index,measured as post-habituation response relative to pre-habituationresponse, was used to evaluate discrimination between crab andeach of the other three mixtures. The habituation index wassignificantly greater for crab (90%) than for oyster (49%) andfor mullet (47%) mixtures. The habituation index for shrimpmixture (65%) was intermediate to these three mixtures. Thus,shrimp mixture is perceived by lobsters as being more similarto crab mixture than is either oyster or mullet mixture. Thesepatterns of discrimination parallel those reported for associativelyconditioned lobsters (Fine-Levy et al., 1987, 1988) and fora population of olfactory receptor cells (Girardot and Derby,1988).