[Three-dimensional architecture of intraosseous vascular anatomy of the hamate: a micro-computed tomography study].

OBJECTIVE: To obtain three-dimensional intraosseous artery of the hamate and to provide the vascular anatomy basis of hamate fracture fixation. METHODS: PbO (lead monoxide, Sinopharm Chemical Reagent Beijing Co. Ltd) was ground into particles less than 40 µm and suspended in turpentine oil (Chemical Reagent Beijing Co. Ltd) at ratios of 1 g : 1.5 mL, 1 g : 1 mL and 1 g : 0.5 mL. Three specimens were investigated. Brachial arteries were cannulated and perfused with lead-based contrast agent. Hamates were harvested and scanned using micro-computed tomography (microCT). The acquisition protocols were as follows: CT scan setup: total rotation [Degrees], 360; rotation steps, 360; X-ray detector setup: transaxial, 2048; axial, 2048; exposure time, 1 500 ms, Binning, 1; system magnification: high-med. X-ray tube setup: 80 kV, 500 mA current. The down-sampling factor used in the reconstruction was 2. The effective voxel size of the final image was 27.30 µm. The three-dimensional model of the hamate was generated and the distribution and pattern of vessels were evaluated. RESULTS: There were abundant extraosseous vessels around the hamate. They were mainly running in the tendons and ligaments around the hamate. Four vascular zones were identified on the hamate surface. They were on the palmar platform of the hamate body, on the dorsal side, on the ulnar side and on the tip of hamulus, namely. There were anastomoses among 4 vascular zones. We did not observe any vessels penetrating through the articular cartilage. The extraosseous vessels of the vascular zones gave a number of intraosseous branches into the hamate. The hamate body received intraosseous blood supply from the dorsal, palmar and ulnar while the hamulus from the palmar, ulnar and hamulus tip. There were some intraosseous branches anastomosing with each other. CONCLUSION: The extraosseous and intraosseous vessels of the hamate were more than what used to be considered. The hamate body and hamulus received blood supply from multiple directions and arteries anastomosed extensively both outside and inside the hamate, making it possible that the intraosseous perfusion survived after fracture. It is likely that the nonunion after the hamate fracture is not caused by the vascular damage but the malalignment of the fragments.

Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis.

We have identified a second form of the type-II neuropeptide encoding a molt inhibiting hormone-like (MeeMIH-B) neuropeptide. MeeMIH-B showed only a 70% amino acid identity to the MIH-A (formerly MIH) isolated from the same species, suggesting a possible different function of the deduced neuropeptide. Like other neuropeptide members of the CHH family, the MIH-B gene consists of three exons separated by two introns. The levels of MIH-B mRNA transcript in the eyestalk decrease in the initial phase of gonad maturation and increase towards the end of maturation. The drop in MIH-B level suggests an inhibitory role for this neuropeptide in the initiation of vitellogenesis. MIH-B transcripts can also be detected in the brain, thoracic ganglion and ventral nerve cord. Together with the CHH-B peptide that we have previously described, this is the second peptide of the CHH family that can also be identified in the ventral nerve cord and in the XOSG complex. A recombinant MIH-B was produced and a polyclonal antibody against rMIH-B was subsequently generated. Specific anti-rMIH-B antiserum recognized the presence of MIH-B in the sinus gland, X-organs, as well as a giant neuron of the ventral nerve cord. Injection of rMIH-B delayed the molting cycle of the maturing female. Taken together, the results of this study suggest that a drop in MIH-B level may be required for the delay in the molting of the maturing females.

Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibody production, immunocytochemical study and biological assay.

Molting in shrimp is controlled by the molt-inhibiting hormone (MIH) and ecdysone. MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in molt suppression; it is a neuropeptide member belonging to the eyestalk CHH/MIH/GIH family. The cloning of MIH (formerly MIH-like) of the shrimp Metapenaeus ensis has been reported in a previous study. To obtain a large quantity of fusion protein for antibody production and biological assay, the cDNA encoding the shrimp MIH was inserted into the pRSET bacterial expression vector. His-tagged fusion protein was produced and purified by an Ni2+-charged affinity column. Polyclonal antibody to rMIH was subsequently obtained by immunizing rabbits with purified recombinant proteins. Results from Western blot analysis indicated that the antibody was specific. Furthermore, results from immunocytochemical analysis showed that specific cells in three different clusters of the X-organ, the sinus gland and the axonal tract of the eyestalk contain MIH. To test for the molt-inhibiting activity of rMIH, shrimp at intermolt stage were injected with rMIH and the molt cycle duration of the injected shrimp was monitored. A significant increase in molt cycle duration was recorded for the shrimp injected with the recombinant protein.

Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins.

The crustacean eyestalk CHH/MIH/GIH neurohormone gene family represents a unique group of neuropeptides identified mainly in crustaceans. In this study, we report the cloning and characterization of the cDNA and the gene encoding the hyperglycemic hormone (MeCHH-B) of the shrimp Metapenaeus ensis. The amino acid sequence of MeCHH-B shows 85% identity to that of MeCHH-A (formerly MeCHH-like neuropeptide). Two separate but identical MeCHH-B genes were identified in the genome of shrimp by library screening and they are located on different CHH gene clusters. The organization of the MeCHH-B gene is identical to other members of the CHH/MIH/GIH neurohormone family. MeCHH-B is expressed at a constant level in the eyestalks of juveniles and mature females. Unlike the MeCHH-A gene, a low level of MeCHH-B transcripts can also be detected in the central nervous system. Interestingly, the expression pattern of MeCHH-B in the eyestalk of vitellogenic females is reversed to that of the MeCHH-A gene. At the middle stage of gonad maturation, a minimum level of MeCHH-B transcript was recorded and a maximum level of MeCHH-A transcript was detected. Recombinant proteins for MeCHH-A and MeCHH-B were produced by a bacterial expression system. The hemolymph glucose level of bilaterally eyestalk-ablated shrimp increased two-fold 1 h after the rCHH injection and then returned to normal after 2 h. The hyperglycemic effect of these fusion proteins is comparable to that of de-stalked shrimp injected with crude extract from a single sinus gland.

Cloning of a cDNA encoding a putative molt-inhibiting hormone from the eyestalk of the sand shrimp Metapenaeus ensis.

Degenerate primers were designed from the amino acid sequence of the neuropeptide Pej-SGP-IV of the shrimp Penaeus japonicus. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using eyestalk complementary DNA of the sand shrimp Metapenaeus ensis. A partial cDNA that codes for a protein homologous to the neuropeptide Pej-SG-IV was cloned. The partial cDNA was used as a probe to screen the eyestalk cDNA library. Several cDNA clones with nucleotide sequence identical to the partial cDNA were isolated. The largest cDNA is 957 bp with an open reading frame consisting of a coding sequence 315 bp in length. The deduced amino acid of the neuropeptide consists of 77 amino acids and is preceded by a signal peptide of 28 amino acids. Because the deduced amino acid sequence of the shrimp cDNA is highly homologous to the Pej-SGP-IV of P. japonicus (which is molt inhibiting) and to other crustaceans' molt-inhibiting hormones (MIHs), the shrimp neuropeptide is tentatively called MeMIH. Northern blot analysis and RT-PCR showed that MeMIH is expressed in the postmolt, intermolt, and premolt stages of the shrimp eyestalks and the brain. Moreover, RNA message can also be detected in the nervous tissues of newly developed larvae. MeMIH is, however, not found in the muscle, swimming leg, and hepatopancreas. Results from genomic Southern blot analysis and amplification of the shrimp genomic DNA by polymerase chain reaction (PCR) suggest that a single copy of the MIH gene is present in the genome. The structural organization of the gene for the shrimp putative MIH is similar to that of the crab Charybdis feriatus.

The shrimp hyperglycemic hormone-like neuropeptide is encoded by multiple copies of genes arranged in a cluster.

The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of glucose metabolism. We have cloned and sequenced several cDNAs encoding the preproCHH-like of the shrimp, Metapenaeus ensis. The preproCHH-like peptide of the shrimp consists of a signal peptide, a CHH precursor-like peptide (CPRP) and the CHH-like peptide. Comparative analysis revealed that the signal peptide and the CPRP of the shrimp peptide are the shortest among all the CHHs reported. MeCHH-like is expressed in the eyestalk, but it is not expressed in the heart, hepatopancreas, muscle, nerve cord and pre-hatch embryo. To study the structural organization of the shrimp CHH-like gene, we have screened the genomic DNA library constructed from one shrimp. Three groups of overlapping genomic clones have been isolated. The results from both genomic Southern blot analysis and library screening indicate that the shrimp genome contains at least six copies of the CHH-like genes arranged in a cluster on the chromosome. The size of the CHH-like genes is 1.5-2.1 kb. DNA sequence determinations indicate that the CHH-like genes share 98-100% amino acid sequence identity. There are three exons and two introns in each CHH-like gene. The first intron separates the signal peptide and the second intron separates the mature peptide in the coding region. The 150-200 bp of the upstream 5' flanking region of the CHH-like genes contains promoters with characteristics similar to most eukaryotic genes. Several putative cis-acting elements are also identified in the first 400 bp 5' end upstream region. The organization of the shrimp CHH-like genes is similar to that of the molt inhibiting hormone gene of the same shrimp and the crab, Charybdis feriatus.

PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus).

A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.