Histology of dart tag insertion sites in the epaulette shark

Samples of dermal and epidermal tissues of epaulette sharks Hemiscyllium ocellatum were examined histologically to assess damage caused by tagging. Tissues from around tag sites were removed at time intervals ranging from 100 min to 284 days post-tagging. These samples showed acute and chronic responses to tagging. Acute responses consisted of localized tissue breakdown and haemorrhaging, and occurred within the first few hours after tag insertion. At 10 h post-tagging, an intermediate response was apparent. This phase was characterized by further haemorrhaging and red and white blood cell movement into the wound area. The chronic response observed in the 10–284-day post-tagging samples was characterized by fibrous tissue formation to sequester the tag. This tissue presumably protects the adjacent musculature from further trauma produced by movement of the tag and provides a continuous barrier between the internal and external milieu. Tissue repair appeared to progress consistently in all specimens and no secondary infections at the tag site were seen. Tagging produced only localized tissue disruption and did not appear to be detrimental to the long term health of individual sharks. Our findings show that spaghetti style dart tagging is an acceptable method for marking individuals (40–75+ cm total length) of this species.     

Analysis of tissue responses to fin tagging in Australian carcharhinids

Tissue responses to the application of Rototags and Jumbo Rototags in the first dorsal fin of Carcharhinus melanopterus, C. obscurus and C. plumbeus were examined. The acute response included tissue tearing and haemorrhage and was present by 5 days post-tagging. The intermediate response had begun by 20 days post-tagging and continued beyond 207 days. This response involved decreased red blood cell activity as the inflammatory response commenced. The chronic response had begun by 301 days and was complete by 553 days with a layer of fibrous connective tissue walling off the tag. External damage to the fin was caused by continued abrasion by the tag. Repair scales were observed at 242 days using scanning electron microscopy and were confirmed histologically in 61- and 553-day samples. Repair scales were not seen in areas of continuous abrasion. No infection was observed in tissues surrounding the wound. Disruption of the fin surface was observed due to abrasion by the tag, but did not appear to cause a severe tissue reaction. The tissue responses observed were consistent with a normal, but relatively slow, healing in the vicinity of the tag wound. Use of Rototags or Jumbo Rototags appears to be an efficient way of marking elasmobranchs with minimal damage to the shark.     

Cellular defense reactions of insect hemocytes in vivo: Nodule formation and development in Galleria mellonella and Pieris brassicae larvae

Galleria mellonella and Pieris brassicae larvae were injected with a standardized dose of killed Bacillus cereus and other bacteria and the reactions of hemocytes followed in the first 24 hr by dissection and histology. Nodules formed in all insects injected with nonpathogens, but a pathogen, Staphylococcus aureus, failed to provoke this reaction. Within 5 min, clumps consisting of granular hemocytes, plasmatocytes, and bacteria were found attached to the internal surfaces of the insects. In the following hours, the cells comprising the clumps broke down and merged with a melanizing acellular substance, and the necrosing masses became encapsulated by plasmatocytes to form mature nodules. The role of granular hemocytes in the formation of the initial cell/bacteria aggregates is discussed along with the possible importance of nodules to the cellular defense reactions of insects.     

Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis.     

Granular phenoloxidase involved in cuticular melanization in the tobacco hornworm: regulation of its synthesis in the epidermis by juvenile hormone

The granular phenoloxidase (PO) that is responsible for cuticular melanization in Manduca sexta larva was purified and an antibody was prepared. This granular PO was found to consist of four isozymes of 90 kDa with isoelectric points ranging from 5.7 to 5.85. The enzyme was immunologically and electrophoretically distinct from the cuticular wound PO, a second cuticular PO common to all larval cuticle, and the hemolymph PO. Both [14C]mannose and [14C]sialic acid were incorporated into the granular PO, showing that this granular PO was a glycoprotein whose sugar moiety was a complex oligosaccharide. When no juvenile hormone (JH) was present at the head capsule slippage (HCS) stage, the epidermis began synthesizing PO 6 hr later. This epidermal synthesis was maximal 12 hr after HCS at which time the PO appeared in the cuticle, and then synthesis declined. When synthesis ceased about 23 hr after HCS, no further incorporation into the cuticle was observed. As melanization proceeded, immunologically detectable cuticular PO decreased. Application of 0.1 microgram JH I at the time of HCS inhibited synthesis of PO by the epidermis and thus prevented melanization. JH application after PO synthesis had begun (8 hr after HCS) prevented its subsequent synthesis, causing partial melanization. Thus, the absence of JH is necessary during the period of epidermal synthesis of the granular PO to allow complete melanization.     

Time course and localization of DNA synthesis during wound healing of potato tuber tissue

DNA synthesis is induced in potato tuber tissue by wounding and starts after a lag period of about 8 hr. As demonstrated by the incorporation of labeled precursors, it reaches its peak between 14 and 18 hr after cutting, and returns to the initial low level before 24 hr, the time of first cell divisions. DNA synthesis is confined to those 2 or 3 cell layers below the wound, where cell division and starch degradation are observed later. Protein synthesis and increase in respiration extend much deeper into the tissue. Both the time course of DNA synthesis and its spatial distribution show patterns different from those of other wound-induced metabolic activities. As wound healing in potato tuber tissue involves the establishment of specific patterns with respect to the time course of induced metabolic activities as well as their spatial distribution, it should be considered and studied as a developmental process. The practical advantages of the system are discussed.     

Ultrastructure of the melanization response of Aedes trivittatus against inoculated Dirofilaria immitis microfilariae

Scanning and transmission electron microscopy revealed that intrathoracically-inoculated microfilariae (mff) of Dirofilaria immitis elicited a rapid and effective immune response in the hemocoel of Aedes trivittatus mosquitoes. Hemocyte lysis and melanization of inoculated mff began immediately following exposure to the hemolymph environment. Initial melanin accumulation occurred at any site along the surface of mff and rapidly increased in thickness. Hemocyte encapsulation generally described for insects did not occur, but hemocytes might be necessary for activation of the melanization response. Although intact hemocytes were never abundant, those that were present seemed to show an active secretion of membrane-bound vacuoles directed toward mff. Activated hemocytes were in close association, but never in direct contact with the parasite, and were most commonly seen in various stages of lysis. Numerous cell remnants were noted throughout the developing melanin capsule. Parasites were completely melanized by 24 hr postinoculation (PI). By about 3 days PI, a membrane began to form around deposited melanin and hemocyte remnants. This developed into a double membrane-like structure of 25-30 nm thickness and resulted in the enclosure and isolation of the mff, melanin deposits, and cellular remnants from hemolymph components. It is suggested that this membrane functions as a boundary to isolate the melanized parasite and prevents additional hemocyte involvement.     

Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368

Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.     

In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae

The Manduca sexta larva-specific immune protein, scolexin, was isolated and (14)CH(3)-labelled by reductive alkylation. The influence of the bacterium Streptococcus faecalis on the hemocoelic distribution of the labelled scolexin was then analyzed. During bacterial challenge, most of the scolexin signal was detected in association with the hemocyte aggregations and nodules which formed; in this respect the protein sometimes appeared to be associated with hemocytes which had phagocytized bacteria, while at other times it was most concentrated in the nodule-associated, and free, coagulum. Areas of high scolexin activity were sometimes detected at various sites on the surface of the fat body. The scolexin did not appear to bind directly to bacterial cells. Up to 24 hr following the injection of S. faecalis, the larvae were still carrying out the formation of nodules; unlike the nodules of the 3 and 6 hr intervals, the nodules observed at 21-24 hr were covered with an apparently humorally derived, coagular capsule.     

Assay of radiation effects in mouse skin as expressed in wound healing

The effect of 150 kVp X irradiation on the healing of full depth surgical wounds in the lower dorsal skin of the mouse was assayed by measuring the wound strength of seven 2-mm-wide segments along each wound. The strength of unirradiated wounds increased with time in two phases: during the first 2 weeks it reached nearly half of the values recorded from unwounded skin, after which the rate of increase slowed for at least 2 weeks before beginning a second increase. By 150 days, the breaking strength of the wound was about 80% of that of unwounded skin. A single dose of 18 Gy prior to wounding reduced the strength of the wounds to about one-third to one-half that of an unirradiated wounds within the 3 months of follow-up. The effect of irradiation on wound strength did not change as the interval between exposure and wounding was increased to 2 months but decreased slightly when this interval was extended to 3 months. When the healing wound was irradiated within 5 days of surgery, the effect on healing was about the same as with preirradiation; if irradiation was delayed for 12 days after wounding the second phase of healing was only postponed and the wound strength ultimately approached the values recorded from unirradiated wounds. The wound strength of skin preirradiated by X rays and assayed 14 days after wounding showed a clear sigmoid dose response with a threshold between 8 and 10 Gy and a plateau at the maximum effect above 20 Gy. The persistence for at least 3 months of the effect of radiation on wound healing suggests that the tissues involved in the healing process are normally proliferating slowly. The accelerated expression of radiation injury through surgical wounding permits the early quantification of the radiation response of tissues that would normally be delayed in their expression of radiation damage.     

毛竹地下茎对创伤的反应

研究了在中国最为广泛栽培的毛竹(Phyllostachys edulis(Cart.)H.de Lehaie)的地下茎的创伤反应。创伤后的第1天,在伤口附近没有明显反应;2d之后,创口附近可以观察到生理反应的代谢物,在后生木质部的导管中以及在基本组织细胞的胞间隙出现粘状物质,这些粘状物质具有果胶特性;创伤后1周,创口附近的筛管及基本组织中的短细胞的细胞壁变成木质化。同时,基本组织中的长细胞的内壁出现新的次生壁的沉积;2周后,创伤反应的组织与未受创伤的组织之间的区别变得更加明显;4周后,一些导管完全充满了粘性物质,但没有观察到侵填体。由于细胞壁的木质化及酚类物质的填充,筛管完全失去了功能。对创伤后6周的材料进行观察的结果表明,其创伤反应的范围不再扩展,在创伤组织与未受伤组织之间的基本组织的细胞壁变得相当厚,从而在两者之间形成一道屏障。毛竹地下茎的这种创伤反应与毛竹竹竿的创伤反应基本上是一致的,只是略有不同。     

The immobility of pectic substances in injured tomato leaves and its bearing on the identity of the wound hormone

It has been suggested that pectic polysaccharides (or oligosaccharides cleaved from them) are liberated from the cell wall upon wounding of leaf tissue, and that they act as long-distance hormones evoking a defence response in neighbouring uninjured leaves (P.D. bishop et al. 1981, Proc. Natl. Acad. Sci. USA 78, 3536–3540, and cited literature). We have tested this hypothesis by infiltration of radioactive pectic fragments (rhamnogalacturonans and homogalacturonans of degrec of polymerisation down to 6) into wounds on tomato leaves. No radioactivity was exported from the treated leaf. [¹⁴C]Sucrose, applied in the same way, was effectively translocated, probably via the phloem. We suggest that pectic substances are not themselves long-distance wound hormones. The possibility remains that pectic substances, solubilised on wounding, act in the immediate vicinity of the wound to stimulate the dispatch of a second messenger, which would be the long-distance wound hormone.Abbreviations DP degree of polymerisation - PI proteinase inhibitor protein - PIIF PI inducing factor - QAE quaternary aminoethyl - TLC thin-layer chromatography     

The distribution of centrosomes in migrating endothelial cells during wound healing in situ.

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 microns from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 microns from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

圆背角无齿蚌血细胞在体内和体外损伤修复过程中的功能

以圆前角无齿蚌为材料,采用外套膜小片的异体移植与加入血细胞的体外培养,研究了血细胞在体风,唯物 修复及珍珠囊构建中的功能。结果表明人工育珠手术后的损伤修复中,血细胞有形成无颗粒细胞层修复伤口、６诱导表皮细胞迁移和再生并形成珍珠囊的功能,而在体外的组织培养中加入血细胞比不加入血细胞的对照组,能更快地诱导上皮细胞迁移和再生,在整个伤面形成２覆盖层,即形成类似珍珠囊结构。本文首次在国内、外报道了用细胞培     

Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding

Epithelial wound healing relies on tissue movements and cell shape changes. Our work shows that, immediately after wounding, there was a dramatic cytoskeleton remodeling consisting of a pulse of actomyosin filaments that assembled in cells around the wound edge and flowed from cell to cell toward the margin of the wound. We show that this actomyosin flow was regulated by Diaphanous and ROCK and that it elicited a wave of apical cell constriction that culminated in the formation of the leading edge actomyosin cable, a structure that is essential for wound closure. Calcium signaling played an important role in this process, as its intracellular concentration increased dramatically immediately after wounding, and down-regulation of transient receptor potential channel M, a stress-activated calcium channel, also impaired the actomyosin flow. Lowering the activity of Gelsolin, a known calcium-activated actin filament–severing protein, also impaired the wound response, indicating that cleaving the existing actin filament network is an important part of the cytoskeleton remodeling process.     

Effects of passive integrated transponder tags on short-term feeding patterns in European perch (Perca fluviatilis)

We tested the feeding behaviour of small European perch (Perca fluviatilis) in a laboratory study during the first 24 h after handling and 23 mm passive integrated transponder (PIT) tag implantation. Feeding commenced almost immediately following tagging and overall feeding patterns were unaffected by tagging. However, untagged perch had more feeding events than PIT-tagged individuals. This discrepancy could be attributed to post-tagging effects or/and reduced room for food due to the presence of the tag in the body cavity.     

Expression of the ipomoelin gene from sweet potato is regulated by dephosphorylated proteins, calcium ion and ethylene

A wound‐inducible cDNA, ipomoelin (IPO) was isolated from the subtraction library of sweet potato (Ipomoea batatas cv. Tainung 57) and used as a molecular probe to investigate the transduction pathway of wounding signal within plant cells. Following mechanical wounding of the leaves of sweet potato, IPO mRNA accumulation peaked at 6 h and then continuously declined. However, IPO gene expression in the apical unwounded leaves began at 6 h after wounding and continued for a further 10 h. Besides mechanical wounding, methyl jasmonate (MeJA) was identified as a signal transducer leading to the accumulation of IPO mRNA. Treatment with salicylic acid reduced the production of IPO mRNA, further supporting the involvement of the octadecanoid pathway in the signal transduction of wounding in sweet potato. In addition, ethylene was involved in the signal pathway and induced the expression of the IPO gene. Furthermore, the application of okadaic acid, a protein phosphatase inhibitor, blocked the accumulation of IPO mRNA induced by MeJA or ethylene, indicating that activation of the IPO gene by both MeJA and ethylene was via dephosphorylated proteins. The presence of a calcium ion chelator or channel blockers also inhibited the expression of the IPO gene after wounding. However, investigation by confocal scanning microscopy further pointed out that mechanical wounding rather than the application of MeJA induced the accumulation of the calcium ion. These results may indicate that the calcium ion is also involved in the activation of IPO mRNA. In addition, wounding signals the accumulation of calcium ion first and then stimulates the biosynthesis of MeJA in sweet potato. Hence, the reaction sequence of signal transducers, including the calcium ion, MeJA and protein kinase/phosphatase, in the wounding signalling pathway of sweet potato is suggested in this report.     

Observations on the phagocytosis and elimination of carmine particles injected into the abdominal musculature of the white shrimp, Penaeus setiferus

White shrimp, Penaeus setiferus, were injected in the abdominal musculature with 0.03 ml of a 1.4% carmine-saline solution and were kept at a temperature of 26°–28°C and a salinity of 23–26‰. Tissue samples were taken at hourly intervals of 1, 6, 12, 18, 24, 30, 36, 42, and 72 hr and at 4, 6, 8, 10, 12, 16, 19, and 33 days post-injection and were examined histologically to determine the sites of phagocytosis and elimination of foreign particulate matter. Within 1 hr post-injection, extracellular clumps of the carmine particles were formed in the hemolymph. These clumps had been invaded by hemocytes at 18 hr, but they persisted throughout the study. Phagocytosis of the particles was accomplished by hemocytes circulating in the hemolymph and by fixed phagocytes in the gill, heart, loose connective tissue, and blood sinusoids in the abdomen. The fate of some phagocytized carmine was climination by the migration of hemocytes through the epithelium of the gills, gut, hepatopancreas, and through the extremities of the pereiopods and the pleopods. Encapsulations or brown nodules were formed in the musculature of the pereiopods around necrotic hemocytes that had phagocytized carmine. A large blister or cyst filled with carmine was formed in the gill cover of one specimen. The obsevation of carmine particles at 33 days post-injection indicates a slow clearance rate of large amounts of abiotic particulate matter in penaeid shrimp.