The G protein-coupled receptor neuropeptide receptor-15 modulates larval development via the transforming growth factor-ß DAF-7 protein in Caenorhabditis elegans.

G protein-coupled receptors (GPCRs) are a major class of membrane receptors that modulate a wide range of physiological functions. These receptors transmit extracellular signals, including secreted bioactive peptides, to intracellular signaling pathways. The nematode Caenorhabditis elegans has FMRFamide-like peptides, which are one of the most diverse neuropeptide families, some of which modulate larval development through GPCRs. In this study, we identified the GPCR neuropeptide receptor (NPR)-15, which modulates C. elegans larval development. Our molecular genetic analyses indicated the following: 1) NPR-15 mainly functions in ASI neurons, which predominantly regulate larval development, 2) NPR-15 interacts with GPA-4, a C. elegans Gα subunit, and 3) NPR-15, along with GPA-4, modulates larval development by regulating the production and secretion of the transforming growth factor-ß (TGF-ß)-like protein DAF-7. The present study is the first report to demonstrate the importance of a GPCR to the direct regulation of a TGF-ß-like protein.

The FMRFamide-like peptide FLP-1 modulates larval development by regulating the production and secretion of the insulin-like peptide DAF-28 in Caenorhabditis elegans.

The FMRFamide-like peptides (FLPs) are conserved in both free-living and parasitic nematodes. This molecular genetic study verified the relevance of the flp-1 gene, which is conserved in many nematode species, to the larval development of the free-living soil nematode Caenorhabditis elegans. Using C. elegans as a model, we found that: (1) FLP-1 suppressed larval development, resulting in diapause; (2) the secretion of FLP-1, which is produced in AVK head neurons, was suppressed by the presence of food (Escherichia coli) as an environmental factor to continue larval development; (3) the FLP-1 reduced the production and secretion of DAF-28, which is produced in ASI head neurons and is the predominant insulin-like peptide (INS) present. FLP-1 is conserved in many species of plant-parasitic root-knot nematodes that cause severe damage to crops. Therefore, our findings may provide insight into the development of new nematicides that can disturb their infection and development.

The FMRFamide-like peptide FLP-2 is involved in the modulation of larval development and adult lifespan by regulating the secretion of the insulin-like peptide INS-35 in Caenorhabditis elegans.

In the animal kingdom, neuropeptides regulate diverse physiological functions. In invertebrates, FMRFamide and its related peptides, a family of neuropeptides, play an important role as neurotransmitters. The FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families and are conserved in nematodes. Our screen for flp genes of the free-living soil nematode Caenorhabditis elegans revealed that the flp-2 gene is involved in the larval development. The gene is also conserved in plant-parasitic root-knot nematodes. Our molecular genetic analyses of the C. elegans flp-2 gene demonstrated as follows: (1) the production and secretion of FLP-2, produced in the head neurons, are controlled by environmental factors (growth density and food); (2) the FLP-2 is involved in not only larval development but also adult lifespan by regulating the secretion of one of the insulin-like peptides INS-35, produced in the intestine. These findings provide new insight into the development of new nematicides.

Intracellular delivery and photothermal therapeutic effects of polyhistidine peptide-modified gold nanoparticles.

Gold nanoparticles (AuNPs) are widely used as an agent in photothermal therapy (PTT) against various cancers. However, a drug delivery system (DDS) is required for effective PTT using AuNPs as AuNPs accumulate passively in tumors. In the present study, we used polyhistidine peptide, a novel cell-penetrating peptide, which is efficiently internalized into tumor cells, as a DDS carrier for PTT using AuNPs. Polyhistidine peptide-modified AuNPs are efficiently internalized into RERF-LC-AI human lung squamous cancer cells and localized to the intracellular lysosome, which is based on the nature of the polyhistidine peptide. Furthermore, the polyhistidine peptide-modified AuNPs inhibited proliferation of RERF-LC-AI cells in a polyhistidine peptide modification-dependent manner under 660 nm laser irradiation. Quantitative real-time PCR showed increased expression levels of an apoptosis-related gene (bax) and heat stress-related gene (hsp70) in RERF-LC-AI cells treated with polyhistidine peptide-modified AuNPs and laser. Our findings highlight the efficacy of AuNPs modified with H16 peptide in PTT.

In vitro transcytosis of Helicobacter pylori histidine-rich protein through gastric epithelial-like cells and the blood-brain barrier.

Recent epidemiological studies have supported the correlation between Helicobacter pylori infection and the development of Alzheimer's disease. HpHpn, a histidine-rich H. pylori protein, forms amyloid-like oligomers; it may be a pathogenic factor for Alzheimer's disease progression. HpHpn may also be transported from the gastric epithelium to the brain. However, HpHpn is secreted from H. pylori on the outer surface of gastric epithelia; therefore, the hypothesized movement of HpHpn across the gastric epithelium to the blood remains controversial. Here, we found the HpHpn showed acidic pH-dependent cellular uptake and subsequent secretion in human gastric epithelial-like carcinoma cells. Furthermore, HpHpn exhibited in vitro permeability across the blood-brain barrier. Although further in vivo experiments are required, our findings suggest that in vitro transcytosis of HpHpn in gastric epithelial cells and the blood-brain barrier may provide new insights into the correlation between H. pylori infections and Alzheimer's disease progression.

Dityrosine Crosslinking of Collagen and Amyloid-ß Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans.

(1) Background: Vitamin B12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid ß, we found that vitamin B12 deficiency did not affect the gene and protein expressions of amyloid ß but increased the formation of dityrosine crosslinking in the amyloid ß protein. (3) Conclusions: Vitamin B12-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid ß formation, which might promote its stabilization and toxic oligomerization.

Direct protein delivery into intact plant cells using polyhistidine peptides.

Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8-His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

Biopsy remains indispensable for evaluating bone marrow involvement in DLBCL patients despite the use of positron emission tomography.

Initial staging by positron emission tomography/computed tomography (PET/CT) scanning is recommended for patients with diffuse large B-cell lymphoma (DLBCL). Whether both PET/CT and bone marrow biopsy (BMB) are required remains unclear. This study examined whether staging by PET/CT is sufficient. Participants with untreated DLBCL assessed using both PET/CT and BMB were included. Patients received independent diagnostic assessments from a radiologist and a hematopathologist. Both hematoxylin-eosin staining and CD20 immunostaining were performed to determine the bone marrow involvement in BMB. A total of 84 patients were included. The number of patients with positive bone marrow involvement identified by PET/CT and BMB was 16 (19%) and 22 (26%), respectively. Eight (10%) patients showed positive results in both tests. When considering BMB as a reference, PET/CT showed 36% sensitivity and 87% specificity, with positive and negative predictive values of 50% and 79%, respectively. BMB-positive patients had shorter progression-free (PFS) and overall (OS) survival than their BMB-negative counterparts. Compared to PET/CT-negative patients, patients with positive results did not show any significant differences in PFS and OS. However, among 16 PET/CT-positive patients, poor PFS and OS were observed among patients who were also BMB positive. BMB remains a mandatory step in staging of untreated DLBCL patients.

Comparison of 18F-FDG PET/CT and 67Ga-SPECT for the diagnosis of fever of unknown origin: a multicenter prospective study in Japan.

OBJECTIVE: The aim of this multicenter prospective study was to compare the sensitivity of 18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT) with that of 67Ga single photon emission computed tomography (SPECT) for the identification of the site of greatest importance for the final diagnosis of the cause of fever of unknown origin (FUO). METHODS: The study participants consisted of patients with an axillary temperature ≥ 38.0 °C on ≥ 2 occasions within 1 week, with repeated episodes for ≥ 2 weeks prior to providing consent, and whose final diagnosis after undergoing specific examinations, including a chest-to-abdomen CT scan, was uncertain. All the patients underwent FDG-PET/CT imaging first, followed by 67Ga-SPECT imaging within 3 days. The results of the FDG-PET/CT and 67Ga-SPECT examinations were reviewed by the central image interpretation committee (CIIC), which was blinded to all other clinical information. The sensitivities of FDG-PET/CT and 67Ga-SPECT were then evaluated with regard to identifying the site of greatest importance for a final diagnosis of the cause of the fever as decided by the patient's attending physician. The clinical impacts (four grades) of FDG-PET/CT and 67Ga-SPECT on the final diagnosis were evaluated. RESULTS: A total of 149 subjects were enrolled in this study between October 2014 and September 2017. No adverse events were identified among the enrolled subjects. Twenty-one subjects were excluded from the study because of deviations from the study protocol. Among the 128 remaining subjects, a final diagnosis of the disease leading to the appearance of FUO was made for 92 (71.9%) subjects. The final diagnoses in these 92 cases were classified into four groups: noninfectious inflammatory disease (52 cases); infectious disease (31 cases), malignancy (six cases); and other (three cases). These 92 subjects were eligible for inclusion in the study's analysis, but one case did not meet the PET/CT image acquisition criteria; thus, PET/CT results were analyzed for 91 cases. According to the patient-based assessments, the sensitivity of FDG-PET/CT (45%, 95% CI 33.1-58.2%) was significantly higher than that for 67Ga-SPECT (25%, 95% CI 15.5-37.5%) (P = 0.0029). The clinical impact of FDG-PET/CT (91%) was also significantly higher than that for 67Ga-SPECT (57%, P < 0.001). CONCLUSIONS: FDG-PET/CT showed a superior sensitivity to 67Ga-SPECT for the identification of the site of greatest importance for the final diagnosis of the cause of FUO.

A polylysine-polyhistidine fusion peptide for lysosome-targeted protein delivery.

Cell-penetrating peptides (CPPs) can deliver payloads into cells by forming complexes with bioactive molecules via either covalent or non-covalent bonds. Previously, we reported polyhistidine (H16 peptide: HHHHHHHHHHHHHHHH-NH2) as a new CPP. This peptide is anticipated to be a valuable new carrier for drug delivery to intracellular lysosomes; the peptide can transport macromolecules into these organelles. In the present study, we examined the application of the H16 peptide as a drug delivery system (DDS) to reverse to lysosomal storage disease (LSD) in cells in vitro. LSDs are metabolic disorders caused by the loss of specific lysosomal enzymes. The majority of lysosomal enzymes are acidic proteins and we utilized this common feature for our DDS. We synthesized a polylysine-polyhistidine fusion peptide (K10H16 peptide: KKKKKKKKKKGHHHHHHHHHHHHHHHH-NH2) and developed a simple method for transporting acidic proteins into intracellular lysosomes via formation of complexes of enzymes with the K10H16 peptide by electrostatic interaction. First, we demonstrated our strategy using maltose-binding protein-fused green fluorescent protein (MBP-GFP) to model an acidic protein. The K10H16 peptide bound to MBP-GFP and transported it into intracellular lysosomes. Further, alpha-galactosidase A (GLA), one of the lysosomal enzymes associated with LSD, was also delivered to intracellular lysosomes by the peptide. The complex between K10H16 peptide and GLA restored typical proliferation to LSD cells, which otherwise grew more slowly than normal cells. These results suggest that K10H16 peptide replenished lysosomal enzyme deficiency in LSD cells. The K10H16 peptide may be useful as a DDS for LSD therapy.

Involvement of Spermidine in the Reduced Lifespan of Caenorhabditis elegans During Vitamin B12 Deficiency.

Vitamin B12 deficiency leads to various symptoms such as neuropathy, growth retardation, and infertility. Vitamin B12 functions as a coenzyme for two enzymes involved in amino acid metabolisms. However, there is limited information available on whether amino acid disorders caused by vitamin B12 deficiency induce such symptoms. First, free amino acid levels were determined in vitamin B12-deficient Caenorhabditis elegans to clarify the mechanisms underlying the symptoms caused by vitamin B12 deficiency. Various amino acids (valine, leucine, isoleucine, methionine, and cystathionine, among others) metabolized by vitamin B12-dependent enzymes were found to be significantly changed during conditions of B12 deficiency, which indirectly affected certain amino acids metabolized by vitamin B12-independent enzymes. For example, ornithine was significantly increased during vitamin B12 deficiency, which also significantly increased arginase activity. The accumulation of ornithine during vitamin B12 deficiency constitutes the first report. In addition, the biosynthesis of spermidine from ornithine was significantly decreased during vitamin B12 deficiency, likely due to the reduction of S-adenosylmethionine as a substrate for S-adenosylmethionine decarboxylase, which catalyzes the formation of spermidine. Moreover, vitamin B12 deficiency also demonstrated a significant reduction in worm lifespan, which was partially recovered by the addition of spermidine. Collectively, our findings suggest that decreased spermidine is one factor responsible for reduced lifespan in vitamin B12-deficient worms.

Development of Organelle Replacement Therapy Using a Stearyl-Polyhistidine Peptide against Lysosomal Storage Disease Cells.

We previously reported on a polyhistidine peptide, His16 peptide, as a new cell-penetrating peptide. This peptide is anticipated to be a new carrier for drug delivery systems (DDSs) for targeting intracellular lysosomes because it can transport macromolecules (e.g., liposomes) into these organelles. In the present study, we examined the application of His16 peptide as a DDS carrier against lysosomal storage disease (LSD) cells. LSDs are metabolic disorders caused by loss of specific lysosomal enzymes. For the treatment of LSD cells, we devised a system designated organelle replacement therapy (ORT). ORT is a strategy for transporting exogenous lysosomes containing all kinds of lysosomal enzymes from normal cells into endogenous lysosomes in LSD cells using His16 peptide. To develop the ORT system, we prepared His16 peptide-modified healthy lysosomes (His16-Lyso) by insertion of a stearyl-His16 peptide into a hydrophobic region in the lysosomal membrane. His16-Lyso showed cellular uptake and localization to endogenous lysosomes in LSD cells. His16-Lyso also restored the proliferation of LSD cells, which otherwise showed slower proliferation than normal cells. These results suggested that His16-Lyso replenished deficient lysosomal enzymes in LSD cells. The results further suggest that His16-Lyso are promising candidates as a treatment tool for LSD cells and to establish a foundation for ORT.

Drug delivery using polyhistidine peptide-modified liposomes that target endogenous lysosome.

Cell-penetrating peptides (CPPs) can deliver payloads into cells by forming complexes with bioactive molecules via covalent or non-covalent bonds. Various CPPs have been applied in CPP-modified liposomes, and their effectiveness is highly regarded in liposomal drug delivery systems (DDSs). Previously, we have reported on the polyhistidine peptide (H16 peptide: HHHHHHHHHHHHHHHH-NH2) as a new CPP. The H16 peptide has a higher cell-penetrating capacity than well-known CPPs and delivers small molecules such as fluorescent dyes, bioactive peptides, and proteins into mammalian cells. However, it is not known whether the H16 peptide can deliver large cargos such as liposomes into cells. To assess the potential of the H16 peptide, in this study, we developed H16 peptide-modified liposomes (H16-Lipo) and evaluated their effectiveness in a liposomal DDS. The H16-Lipo was prepared by inserting a stearyl-H16 peptide into the hydrophobic region of a liposome. The H16-Lipo was internalized into human fibrosarcoma cells via multiple endocytosis pathways and localized to intracellular lysosomes. Based on this result, we used the H16-Lipo as a lysosome-targeting DDS. The H16-Lipo delivered alpha-galactosidase A (GLA), one of the lysosomal enzymes, to intracellular lysosomes and improved the proliferation of GLA-knockdown cells. These results suggest that the H16-Lipo is an effective drug carrier for lysosomal enzymes in a lysosome-targeting DDS. The loss of lysosomal enzymes has been known to induce metabolic disorders, called lysosomal storage diseases (LSDs). Our findings indicate that this combination of the H16 peptide and a liposome is a promising candidate as a DDS for the treatment of LSDs.

Comparison of physiological functions of antagonistic insulin-like peptides, INS-23 and INS-18, in Caenorhabditis elegans.

In Caenorhabditis elgans, insulin-like peptides have significant roles in modulating larval diapause and adult lifespan via the insulin/IGF-1 signaling (IIS) pathway. Although 40 insulin-like peptides (ILPs) have been identified, it remains unknown how ILPs act as either agonists or antagonists for their sole receptor, DAF-2. Here we found 1) INS-23 functions as an antagonistic ILP to promote larval diapause through the IIS pathway like a DAF-2 antagonist, INS-18, 2) INS-23 and INS-18 have similar biochemical functions. In addition, our molecular modeling suggests that INS-23 and INS-18 have characteristic insertions in the B-domain, which are crucial for the recognition of the insulin receptor, when compared with DAF-2 agonists. These characteristic insertions in the B-domain of INS-23 and INS-18 would modulate their intermolecular interactions with the DAF-2 receptor, which may lead these molecules to act as antagonistic ligands. Our study provides new insight into the function and structure of ILPs.

Measurement of serum isoform [-2]proPSA derivatives shows superior accuracy to magnetic resonance imaging in the diagnosis of prostate cancer in patients with a total prostate-specific antigen level of 2-10 ng/ml.

OBJECTIVE: More accurate diagnostic procedures for prostate cancer are needed to avoid unnecessary biopsy due to the low specificity of prostate-specific antigen (PSA). Recent studies showed that the percentage of serum isoform [-2]proPSA (p2PSA) to free PSA (%p2PSA), the Prostate Health Index (PHI) and magnetic resonance imaging (MRI) were more accurate than PSA. The aim of this study was to test the accuracy of %p2PSA, PHI and MRI in discriminating patients with and without prostate cancer. MATERIALS AND METHODS: The subjects were 50 consecutive men with a PSA level of 2.0-10.0 ng/ml, who underwent prostate biopsy from October 2012 to July 2014. These patients underwent multiparametric MRI before biopsy, and their serum samples were measured for PSA, free PSA and p2PSA. The sensitivity, specificity and accuracy of PHI, %p2PSA and MRI were compared with PSA in the diagnosis of biopsy-confirmed prostate cancer. RESULTS: In a univariate analysis, %p2PSA [area under the curve (AUC): 0.811] and PHI (AUC 0.795) were more accurate than MRI (AUC: 0.583) and PSA (AUC: 0.554) for prostate cancer detection. At 60% sensitivity, the specificity of PHI (76.5%) was higher than that of MRI (52.9%). For significant cancer detection, %p2PSA (AUC: 0.745), PHI (AUC: 0.791) and MRI (AUC: 0.739) were marginally more accurate than PSA (AUC: 0.696). At 85% sensitivity, the specificity of MRI (62.1%) was higher than that of PHI (34.5%). CONCLUSION: PHI and %p2PSA can be used for screening the general population and MRI can be used for detection of significant cancer in patients suspected, from screening tests, of having prostate cancer.

Short polyhistidine peptides penetrate effectively into Nicotiana tabacum-cultured cells and Saccharomyces cerevisiae cells.

The polyhistidine peptides (PHPs) have been previously reported as novel cell-penetrating peptides and are efficiently internalized into mammal cells; however, penetration of PHPs into other cell types is unknown. In this study, the cellular uptake of PHPs in plant and yeast cells was found to be dependent on the number of histidines, and short PHPs (H6-H10 peptides) showed effective internalization. The H8 peptide showed the highest cell-penetrating capacity and localized to vacuoles in plant and yeast cells. Low-temperature conditions inhibited significantly the cellular uptake of short PHPs by both cells. However, net charge neutralization of PHPs also completely inhibited cellular uptake by plant cells, but not by yeast cells. These results indicate that short PHPs penetrate effectively into plant and yeast cells by similar mechanism with the exception of net charge dependency. The findings show the short PHPs are promising candidates for new delivery tools into plant and yeast cells.

Vitamin B12 deficiency results in severe oxidative stress, leading to memory retention impairment in Caenorhabditis elegans.

Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B12 deficiency, although the underlying disease mechanisms associated with vitamin B12 deficiency are poorly understood. Vitamin B12 deficiency was found to significantly increase cellular H2O2 and NO content in Caenorhabditis elegans and significantly decrease low molecular antioxidant [reduced glutathione (GSH) and L-ascorbic acid] levels and antioxidant enzyme (superoxide dismutase and catalase) activities, indicating that vitamin B12 deficiency induces severe oxidative stress leading to oxidative damage of various cellular components in worms. An NaCl chemotaxis associative learning assay indicated that vitamin B12 deficiency did not affect learning ability but impaired memory retention ability, which decreased to approximately 58% of the control value. When worms were treated with 1mmol/L GSH, L-ascorbic acid, or vitamin E for three generations during vitamin B12 deficiency, cellular malondialdehyde content as an index of oxidative stress decreased to the control level, but the impairment of memory retention ability was not completely reversed (up to approximately 50%). These results suggest that memory retention impairment formed during vitamin B12 deficiency is partially attributable to oxidative stress.

Pharmacokinetics of single dose radium-223 dichloride (BAY 88-8223) in Japanese patients with castration-resistant prostate cancer and bone metastases.

OBJECTIVE: This open-label, non-randomized, phase I study examined the pharmacokinetics (PK) and radiation dosimetry of a single dose of radium-223 in Japanese patients with castration-resistant prostate cancer (CRPC) and bone metastases. METHODS: Six male Japanese patients (mean age 72.5 years, range 65-79 years) with histologically or cytologically confirmed stage IV adenocarcinoma of the prostate were recruited. A single IV dose of radium-223 was delivered intravenously (IV) via slow bolus over a 2-5 min period: Cohort 1 received 50 kBq/kg and Cohort 2 received 100 kBq/kg. RESULTS: Following IV injection, radium-223 was rapidly eliminated from the blood in a multi-phasic manner. The fraction of the injected activity of radium-223 retained in the whole body 24 h following injection was 85 %. Biodistribution results showed initial bone uptake was 52 % (range 41-57 %). The maximum activity of radium-223 in the bone was observed within 2 h of dosing. Activity of radium-223 passed through the small intestine within 24 h. No activity was detected in other organs. The major radiation dose from radium-223 was found in osteogenic cells; calculated absorbed doses in osteogenic cells and in the red marrow were 0.76 Gy/MBq and 0.09 Gy/MBq, respectively. CONCLUSIONS: In Japanese patients with CRPC and bone metastases, radium-223 (IV) achieved maximum activity in the bone rapidly and passed through the intestine within 24 h, without signs of activity in other organs. The PK profile and absorbed radiation dose in organs and tissues in Japanese patients were similar to data from non-Japanese patients. Trial registration identification: NCT01565746.

Diapause is associated with a change in the polarity of secretion of insulin-like peptides.

The insulin/IGF-1 signalling (IIS) pathway plays an important role in the regulation of larval diapause, the long-lived growth arrest state called dauer arrest, in Caenorhabditis elegans. In this nematode, 40 insulin-like peptides (ILPs) have been identified as putative ligands of the IIS pathway; however, it remains unknown how ILPs modulate larval diapause. Here we show that the secretory polarity of INS-35 and INS-7, which suppress larval diapause, is changed in the intestinal epithelial cells at larval diapause. These ILPs are secreted from the intestine into the body cavity during larval stages. In contrast, they are secreted into the intestinal lumen and degraded during dauer arrest, only to be secreted into the body cavity again when the worms return to developmental growth. The process that determines the secretory polarity of INS-35 and INS-7, thus, has an important role in the modulation of larval diapause.

Cellular uptake and in vivo distribution of polyhistidine peptides.

Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.