Transplantation of adipose tissue-derived microvascular fragments promotes therapy of critical limb ischemia.

BACKGROUND: Adipose tissue-derived microvascular fragments are functional vessel segments derived from arterioles, capillaries, and veins. Microvascular fragments can be used as vascularization units in regenerative medicine and tissue engineering containing microvascular networks. However, the in vivo therapeutic and vascularization properties of human microvascular fragments have not been investigated. METHODS: In this study, we isolated microvascular fragments, stromal vascular fractions, and mesenchymal stem cells from human lipoaspirate and studied their therapeutic efficacy and in vivo vasculogenic activity in a murine model of hindlimb ischemia. In addition, in vivo angiogenic activity and engraftment of microvascular fragments into blood vessels were measured using Matrigel plug assay. RESULTS: Both microvascular fragments and stromal vascular fractions contain not only mesenchymal stem cells but also endothelial progenitor cells. In a Matrigel plug assay, microvascular fragments increased the number of blood vessels containing red blood cells more than mesenchymal stem cells and stromal vascular fractions did. The engraftment of the microvascular fragments transplanted in blood vessels within the Matrigel plug significantly increased compared to the engraftment of mesenchymal stem cells and stromal vascular fractions. Moreover, intramuscular injection of microvascular fragments markedly increased blood flow in the ischemic hindlimbs and alleviated tissue necrosis compared to that of mesenchymal stem cells or stromal vascular fractions. Furthermore, transplanted microvascular fragments formed new blood vessels in ischemic limbs. CONCLUSIONS: These results suggest that microvascular fragments show improved engraftment efficiency and vasculogenic activity in vivo and are highly useful for treating ischemic diseases and in tissue engineering. Adipose tissue-derived microvascular fragments are vascularization units in regenerative medicine and tissue engineering containing microvascular networks. Intramuscular injection of microvascular fragments markedly increased blood flow in the ischemic hindlimbs and alleviated tissue necrosis. The present study suggests that microvascular fragments show improved engraftment efficiency and vasculogenic activity in vivo and are highly useful for treating ischemic diseases and in tissue engineering.

Differential activity of 16K rat prolactin in different organic systems.

The 16K isoform of rat prolactin (16K rPRL) performs multiple functions in various systems including angiogenesis, tumorigenesis, and reproduction. Recently, 16K rPRL has attained prominence as a possible therapeutic target in pathophysiological conditions. However, the integral function and mechanism of 16K rPRL in various systems has not been elucidated. To this end, a transient gain-of-function animal model was adopted. An expression DNA plasmid containing 16K rPRL or rPRL gene was introduced into the muscle of adult mice by direct injection. The mRNA and protein expression levels of 16K rPRL were detected by initial RT-PCR and subsequent Southern blot and western blot, respectively. When the expression vector was introduced, the results were as follows: First, 16K rPRL combined with rPRL reduced angiogenesis in the testis whereas rPRL alone induced angiogenesis. Second, 16K rPRL combined with rPRL reduced WBC proliferation, whereas rPRL alone increased WBC proliferation. Third, 16K rPRL combined with rPRL reduced diestrus, whereas rPRL alone extended diestrus. Fourth, 16K rPRL combined with rPRL unexpectedly increased testosterone (T) levels, whereas rPRL alone did not increase T levels. Taken together, our data suggest that the 16K rPRL isoform performs integral functions in angiogenesis in the testis, WBC proliferation, and reproduction, although the action of 16K rPRL is not always antagonistic.

Structure of the periplasmic copper-binding protein CueP from Salmonella enterica serovar Typhimurium.

CueP was initially identified as a copper-resistance gene in Salmonella enterica serovar Typhimurium, which has evolved to survive in the phagosomes of macrophages. Recently, CueP was determined to be a periplasmic copper-binding protein and has been implicated in the transfer of copper ions to SodCII in the periplasm. In this study, the crystal structure of CueP has been determined, revealing a V-shaped dimeric structure. The conserved cysteine and histidine residues are clustered on the surface of one side of the C-terminal domain, suggesting that this cysteine- and histidine-rich region is related to the function of CueP. LC-MS/MS analysis established the presence of a disulfide bond between Cys96 and Cys176 under aerobic conditions. Subsequent biophysical analyses showed that the CueP protein binds copper and zinc, and the mutation of Cys104 to serine (C104S) dramatically reduced the binding affinity for copper and zinc, suggesting that the cysteine- and histidine-rich cluster is responsible for copper binding. This study provides a structural basis for the participation of CueP in the resistance of the intracellular pathogen Salmonella to copper.

Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana.

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0Å in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.

Crystallization and preliminary X-ray crystallographic analysis of Salmonella Typhimurium CueP.

Salmonella enterica serovar Typhimurium (S. Typhimurium) can survive in the phagosome of macrophages, causing serious medical and veterinary problems. CueP is uniquely found in S. Typhimurium and has been characterized as a major periplasmic copper-binding protein. Although cueP has been identified as being responsible for the copper resistance of the bacterium in vivo, the biochemical role and three-dimensional structure of CueP remain unknown. In this study, CueP from S. Typhimurium was overexpressed and the recombinant protein was purified using Ni-NTA affinity, anion-exchange and gel-filtration chromatographies. The purified CueP protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to 2.5 Šresolution at 100 K. The crystal belonged to space group P2(1)2(1)2(1). To obtain initial phases, selenomethionyl-substituted protein was overproduced and purified. Optimization of crystallization conditions for the selenomethionyl-substituted protein is in progress.

Crystal structure and functional insight of HP0420-homolog from Helicobacter felis.

Helicobacter pylori infect more than half of the world's population and are considered a cause of peptic ulcer disease and gastric cancer. Recently, hypothetical gene HP0421 was identified in H. pylori as a cholesterol alpha-glucosyltransferase, which is required to synthesize cholesteryl glucosides, essential cell wall components of the bacteria. In the same gene-cluster, HP0420 was co-identified, whose function remains unknown. Here we report the crystal structure of HP0420-homolog of H. felis (HF0420) to gain insight into the function of HP0420. The crystal structure, combined with size-exclusion chromatography, reveals that HF0420 adopts a homodimeric hot-dog fold. The crystal structure suggests that HF0420 has enzymatic activity that involves a conserved histidine residue at the end of the central alpha-helix. Subsequent biochemical studies provide clues to the function of HP0420 and HF0420.

Periplasmic domain of CusA in an Escherichia coli Cu+/Ag+ transporter has metal binding sites.

The resistance nodulation division (RND)-type efflux systems are utilized in Gram-negative bacteria to export a variety of substrates. The CusCFBA system is the Cu(+) and Ag(+) efflux system in Escherichia coli, conferring resistance to lethal concentrations of Cu(+) and Ag(+). The periplasmic component, CusB, which is essential for the assembly of the protein complex, has Cu(+) or Ag(+) binding sites. The twelve-span membrane protein CusA is a homotrimeric transporter, and has a relatively large periplasmic domain. Here, we constructed the periplasmic domain of CusA by joining two DNA segments and then successfully expressed and purified the protein. Isothermal titration calorimetry experiments revealed Ag(+) binding sites with Kds of 10(-6)-10(-5) M. Our findings suggest that the metal binding in the periplasmic domain of CusA might play an important role in the function of the efflux pump.

Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli CusB.

Periplasmic membrane-fusion proteins (MFPs) are an essential component of multidrug and metal-efflux pumps in Gram-negative bacteria. However, the functional structure of MFPs remains unclear. CusCFBA, the Cu(I) and Ag(I) efflux system in Escherichia coli, consists of the MFP CusB, the OMF CusC and the RND-type transporter CusA. The MFP CusB bridges the inner membrane RND-type efflux transporter CusA and the outer membrane factor CusC and exhibits substrate-linked conformational changes which distinguish it from other MFP-family members. CusB from E. coli was overexpressed and the recombinant protein was purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified CusB protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 3.1 A at 100 K. The crystal belonged to space group C222.

Changes in reproductive function and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle.

Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species.