TAT 14

Human acidic fibroblast growth factor (haFGF), a neurotrophin-like growth factor in the brain, plays important roles in the development, differentiation and regeneration of brain neurons, which makes it potential to treat Alzheimer's disease (AD). In this study, haFGF(14-154) and TAT-haFGF(14-154) (haFGF(14-154) fused with the cell-penetrating peptide transactivator of transcription protein transduction domain (TAT-PTD)) were intranasally administrated for 5 weeks to investigate the effects on senescence-accelerated mouse prone-8 (SAMP8) mice (a mouse model of AD). Results showed that TAT-PTD could increase the concentration of haFGF in the brain significantly, and TAT-haFGF(14-154) was more effective than haFGF(14-154) in the same dosage (300 μg/kg). Importantly, TAT-haFGF(14-154) improved the learning and memory abilities of SAMP8 mice in the behavioral test, and promoted the function of cholinergic system by measuring the relevant biomarkers (acetylcholine (ACh) level, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities). TAT-haFGF(14-154) also significantly reduced β-amyloid protein(1-42) (Aβ(1-42)) deposits as well as the levels of Aβ soluble forms in the mice brains and prevented the neurons from apoptosis. Besides, the oxidative stress impairment in the brain and serum was also ameliorated. The results suggest that TAT-haFGF(14-154) could attenuate the disease progression of SAMP8 AD mice, and the mechanism is related to the regulation of neurons microenvironment including neurotransmitters, Aβ pathology and oxidative stress.

Aim:Pregnancy is a hypercoagulability state, the aim of this study was to observe the changes of thrombin-antithrombin complex (TAT), thrombomodulin (TM), tissue plasminogen activator-inhibitor complex (tPAI-C) and plasmin-α2-antiplasmin complex (PIC) during pregnancy and establish trimester-specific reference intervals for Chinese healthy pregnant women.Methods:In total 190 Chinese healthy pregnant women (first trimester 59 cases, second trimester 60 cases and third trimester 71 cases) were recruited in North China. TAT, TM, tPAI-C and PIC were processed on Sysmex HISCL 5000 automated chemiluminescence immune detection system. Trimester-specific reference intervals were established with the 2.5th and 97.5th percentile of the distribution.Results:The reference intervals for TAT, TM, tPAI-C, PIC at trimester 1 were 0.40-3.65 ng/mL, 4.85-8.80 TU/mL, 1.75-6.40 ng/mL, 0.25-1.05 μg/mL, respectively. At trimester 2, the reference intervals were 1.65-8.61 ng/mL, 5.70-9.93 TU/mL, 2.91-7.71 ng/mL, 0.33-2.02 μg/mL, respectively. At trimester 3, the reference intervals were 3.16-12.68 ng/mL, 5.50-14.24 TU/mL, 2.70-10.69 ng/mL, 0.24-1.54 μg/mL, respectively.Conclusions:The changes of TAT, TM, tPAI-C, PIC during pregnancy are presented, and trimester-specific reference intervals for healthy pregnant women are described. The levels of TAT, TM, tPAI-C were increased gradually from trimester 1 to trimester 3, while the PIC level remains stable during all trimesters.

99mTc-N2S2-Tat(49-57)-Lys3-bombesin (99mTc-Tat-BN) is a hybrid peptide synthesized and labeled with99mTc by Santos-Cuevas et al. for molecular imaging of tumors expressing gastrin-releasing peptide receptor (GRPR) (1).Bombesin (BN) is an amphibian neuropeptide consisting of 14 amino acids (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) (2, 3), first isolated from frog skin in 1970 (4). The search for its mammalian counterpart led to the discovery of gastrin-releasing peptide (GRP), which consists of 27 amino acids. GRP and BN share an identical C-terminal region (-Trp-Ala-Val-Gly-His-Leu-Met-NH2), which is necessary for receptor binding and signal transduction (5, 6). Besides the release of gastrin, GRP and BN-like peptides also produce a wide range of other biological responses in diverse tissues such as secretion of endocrine and exocrine glands, maintenance of circadian rhythm, regulation of satiety, and contraction of smooth muscles (7). They also act as potential growth factors for both normal and cancer cells (3, 5, 6). There are four members of the BN receptor family, including three mammalian receptors: GRPR (BB2or BRS2; 384 amino acids), neuromedin B receptor (NMBR, BB1, or BRS1; 390 amino acids), and BN-like receptor 3 (BB3, BRS3, or orphan; 399 amino acids) (5, 7, 8); the fourth receptor (BB4) has only been found in amphibians. GRPR is the only well characterized receptor of this family. GRPR is a glycosylated, 7-transmembrane, G-protein-coupled receptor that, upon binding with its ligands, gives rise to a complex cascade of intracellular reactions. It is normally found in non-neuroendocrine tissues of the breast and pancreas and in neuroendocrine cells of the brain, gastrointestinal tract, lung, and prostate (9). Interestingly, GRPR is overexpressed in prostate cancer as well as tumors of breast, lung, pancreas, ovary, kidney, and gastrointestinal tract. It has been reported that GRPR is expressed at a high density in the intraepithelial neoplasia and primary carcinoma of prostate, whereas normal prostate tissue and, in most cases, benign prostate hyperplasia are predominantly GRPR-negative (10-13).GRPR has attracted significant interest as a target for tumor detection, tumor staging, and evaluation of tumor response to therapy (5, 6, 8, 11). A large number of BN derivatives have been developed, and they have been labeled with99mTc,177Lu,67Ga, and111In for single-photon emission computed tomography and with64Cu,68Ga, and18F for positron emission tomography. The published BN derivatives can be generally classified as truncated BN (6-14 or 7-14) or full-length BN (1-14) analogs (1, 10, 12-17). The truncated BN analogs seem to be favorable because they are usually more stable than the full-length tetradecapeptides forin vivoapplications, and they still bind to the GRPR adequately. However, the full-length peptides offer different labeling methods by attachment of functional groups to the amino acid 1 to 6. In many cases, the amino acids on position 13 (Leu) and 14 (Met) have been replaced by unnatural amino acids (cyclohexylalanine (CyHAla) and norleucine (Nle)), and Lys has been placed on position 3 for attachment of radiolabels with reactive esters. Spacers, chelators, or radiometals have also been widely used for conjugation and for favorable kinetics (18). The BN derivatives can also be divided into agonists and antagonists (19-21). By far, most BN derivatives are agonists. Agonists are internalized into and accumulate within cells, and they have been assumed to exhibit higher uptake by cancer cells than antagonists. However, some reports have shown that uptake of antagonists is much higher than that of agonists because antagonists may have stronger binding for the GRPR than agonists (19, 20).An optimal BN-like radiotracer needs to meet several requirements: high affinity for GRPR, with rapid and specific tumor uptake; high hydrophilicity, with preferred renal excretion and low hepatobiliary excretion; and high stability but relatively rapid clearance from blood (6). Despite a large number of published derivatives, a valid comparison among them for the feasibility of tumor imaging is difficult because standardization between studies is lacking. Generally speaking, the majority of the radiotracers have relatively high renal and hepatic uptake, resulting in low tumor/liver and tumor/kidney ratios. In an effort to increase the tumor cell uptake and consequently improve the image contrast of tumors and their metastases, Santos-Cuevas et al. developed a hybrid peptide radiotracer (99mTc-Tat-BN) and compared it with the GRPR agonist99mTc-EDDA/HYNIC-[Lys3]-BNfor thein vitroandin vivouptake kinetics in GRPR-expressing cancer cells and tumor xenografts (1, 22).99mTc-Tat-BN was constructed by linking the HIV Tat-derived peptide (Tat(49-57)) with the GRPR agonist Lys3-BN peptide. Tat(49-57) is a small basic peptide with a sequence of H-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH2, and it has been successfully used for delivering a large variety of cargoes into cells because of its cell-penetrating properties (11).

Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB) due to its large size. A protein transduction domain (PTD) of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R) model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP), which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA) inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic activation.