Glycine blocks the increase in intracellular free Ca2+  due to vasoactive mediators in hepatic parenchymal cells

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE2, which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca2+ concentration ([Ca2+]i) in hepatic parenchymal cells by agonists released during stress, such as with PGE2 and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca2+]i in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE2 caused a dose-dependent increase in [Ca2+]i from basal values of 130 ± 10 to maximal levels of 434 ± 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca2+ dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca2+ from intracellular stores, also partially blocked the increase in [Ca2+]i caused by PGE2, suggesting that intracellular Ca2+ pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca2+ pools are involved in the increase in [Ca2+]i caused by PGE2. Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca2+]i due to PGE2 in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl− was omitted, glycine was much less effective in preventing the increase in [Ca2+]i due to PGE2. Phenylephrine, an α1-type adrenergic receptor agonist, also increased [Ca2+]i, as expected, from 159 ± 20 to 432 ± 43 nM. Glycine also blocked the increase in [Ca2+]i due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca2+]i in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.

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Stimulation of incretin secretion by dietary lipid: is it dose dependent?

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90601.2008 ◽

2009 ◽

Vol 297 (2) ◽

pp. G299-G305 ◽

Cited By ~ 52

Author(s):

Stephanie M. Yoder ◽

Qing Yang ◽

Tammy L. Kindel ◽

Patrick Tso

Keyword(s):

Lipid Content ◽

Cell Types ◽

Distal Portion ◽

Dietary Lipid ◽

Dependent Manner ◽

Incretin Hormones ◽

Sprague Dawley ◽

Dependent Relationship ◽

Dose Dependent

After the ingestion of nutrients, secretion of the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) by the enteroendocrine cells increases rapidly. Previous studies have shown that oral ingestion of fat stimulates secretion of both incretins; however, it is unclear whether there is a dose-dependent relationship between the amount of lipid ingested and the secretion of the hormones in vivo. Recently, we found a higher concentration of the incretin hormones in intestinal lymph than in peripheral or portal plasma. We therefore used the lymph fistula rat model to test for a dose-dependent relationship between the secretion of GIP and GLP-1 and dietary lipid. Under isoflurane anesthesia, the major mesenteric lymphatic duct of male Sprague-Dawley rats was cannulated. Each animal received a single, intraduodenal bolus of saline or varying amounts of the fat emulsion Liposyn II (0.275, 0.55, 1.1, 2.2, and 4.4 kcal). Lymph was continuously collected for 3 h and analyzed for triglyceride, GIP, and GLP-1 content. In response to increasing lipid calories, secretion of triglyceride, GIP, and GLP-1 into lymph increased dose dependently. Interestingly, the response to changes in intraluminal lipid content was greater in GLP-1- than in GIP-secreting cells. The different sensitivities of the two cell types to changes in intestinal lipid support the concept that separate mechanisms may underlie lipid-induced GIP and GLP-1 secretion. Furthermore, we speculate that the increased sensitivity of GLP-1 to intestinal lipid content reflects the hormone's role in the ileal brake reflex. As lipid reaches the distal portion of the gut, GLP-1 is secreted in a dose-dependent manner to reduce intestinal motility and enhance proximal fat absorption.

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Dose-Dependent Sorafenib-Induced Immunosuppression Is Associated with Aberrant NFAT Activation and Expression of PD-1 in T Cells

Cancers ◽

10.3390/cancers11050681 ◽

2019 ◽

Vol 11 (5) ◽

pp. 681 ◽

Cited By ~ 9

Author(s):

Renuka V. Iyer ◽

Orla Maguire ◽

Minhyung Kim ◽

Leslie I. Curtin ◽

Sandra Sexton ◽

...

Keyword(s):

T Cells ◽

Low Dose ◽

Growth Delay ◽

High Dose ◽

Dependent Manner ◽

Sorafenib Treatment ◽

Baseline Activity ◽

Dose Dependent

The multikinase inhibitor sorafenib is the only standard first-line therapy for hepatocellular carcinoma (HCC). Here, we report the dose-dependent effects of sorafenib on the immune response, which is related to nuclear factor of activated T cells 1 (NFAT1) activity. In vitro and in vivo experiments were performed with low and high doses of sorafenib using human T cells and spontaneous developed woodchuck HCC models. In vitro studies demonstrated that following exposure to a high dose of sorafenib the baseline activity of NFAT1 in T cells was significantly increased. In a parallel event, high dose sorafenib resulted in a significant decrease in T cell proliferation and increased the proportion of PD-1 expressing CD8+ T cells with NFAT1 activation. In the in vivo model, smaller tumors were detected in the low-dose sorafenib treated group compared to the placebo and high-dose treated groups. The low-dose sorafenib group showed a significant tumor growth delay with significantly more CD3+ cells in tumor. This study demonstrates that sorafenib has immunomodulatory effects in a dose- and time-dependent manner. Higher dose of sorafenib treatment was associated with immunosuppressive action. This observed effect of sorafenib should be taken into consideration in the selection of optimum starting dose for future trials.

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CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Blood ◽

10.1182/blood-2019-131559 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 404-404

Author(s):

Akinori Yoda ◽

Daisuke Morishita ◽

Akio Mizutani ◽

Yoshihiko Satoh ◽

Yotaro Ochi ◽

...

Keyword(s):

Low Dose ◽

Synthetic Lethality ◽

Sr Proteins ◽

High Dose ◽

Dependent Manner ◽

Employment Equity ◽

Myeloid Neoplasms ◽

Equity Ownership ◽

Dose Dependent

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as 'chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. These SF mutations are largely mutually exclusive and except for ZRSR2 mutations, are invariably heterozygous, showing prominent hotspots, suggesting that mutations have neomorphic functions and might cause a synthetic lethality when they are homozygous or two SFs are mutated. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression, which are dependent of CLK2 expression levels. Thus, CLK family kinases are possible targets of inhibition by small molecules to induce synthetic lethality in SF-mutated MDS/AML and for this purpose, we have recently developed an orally available and highly potent CLK inhibitor, CTX-712 and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and THP1), CTX-712, strongly inhibited phosphorylation of multiple SR proteins including SRSF3, SRSF4, SRSF5, and SRSF6 that bind to SRSF2. To further investigate the efficacy of CTX-712 in vivo, we established 5 AML-derived xenograft (PDX) models, which treated with varying doses of CTX-712. Among these 5 PDX models, SRSF2 mutation was found in only one case, which had a SRSF2 p.P95H, mutation, while others (a subcutaneous and 3 leukemia model) were negative for SRSF2 mutations. The SRSF2-mutated model showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two-week after treatment, tumor volumes (mm3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.028), and 39 ± 39 (high dose, P=0.0014) (N=5 each, mean ± SEM). CTX-712 also significantly improved the survival of PDX #1. Median survivals (days after engraftment) were 34.5 (vehicle) vs. 93.5 (12.5mg/kg, P=0.015) (N=2 each). Interestingly, another leukemic model carrying KRAS, NF1, and TP53 but not SRSF2 mutations also showed a significant reduction of leukemic burden 2 weeks after CTX-712 treatment; leukemic burden after therapy, as measured by frequency of hCD45+cells in PB (%), were 82 ± 2.2 (vehicle), 17 ± 3.6 (low dose, P<0.0001), and 0.89 ± 0.43 (complete remission, high dose, P<0.0001), (N=4 each, mean ± SEM). In the third PDX model with mutations of FLT3, RAD21, RUNX1, and WT1, CTX-712 administration reduced subcutaneous AML tumors in a dose-dependent manner and achieved partial remission (high dose, P=0.0008) (N=6 each). CTX-712 also significantly improved the survival of the PDX #3 model (high dose, P=0.0069) (N=6 each). In PDX #4, leukemic model with mutations of ASXL1, BCOR, and TET2, high dose CTX-712 therapy strongly reduced the leukemic cell burden than vehicle control (P=0.0027), (N=4 each). CTX-712 also significantly improved the survival of this model (P=0.016) (N=5 each). The last AML PDX #5 model with U2AF1, BCOR, DNMT3A, IDH1, KDM6A, RUNX1, and TET2 mutations was refractory for CTX-712 therapy. Overall, 4 out of 5 PDX AML models showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in 2 cases, including an SRSF2-mutated model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML. CTX-712 is currently in clinical phase 1 trials for relapsed and refractory malignancies. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Morishita:Chordia Therapeutics Inc.: Employment, Equity Ownership. Mizutani:Chordia Therapeutics Inc.: Employment, Equity Ownership. Satoh:Chordia Therapeutics Inc: Employment, Equity Ownership. Miyake:Chordia Therapeutics Inc.: Employment, Equity Ownership. Ogawa:Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties.

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Probenecid Inhibits Platelet Responses to Aggregating Agents in Vitro and Has a Synergistic Inhibitory Effect with Penicillin G

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650561 ◽

1996 ◽

Vol 76 (02) ◽

pp. 239-244 ◽

Cited By ~ 6

Author(s):

M A Packham ◽

M L Rand ◽

D W Perry ◽

D H Ruben ◽

R L Kinlough-Rathbone

Keyword(s):

Platelet Activating Factor ◽

Anion Channel ◽

Inhibitory Effect ◽

Penicillin G ◽

Dependent Manner ◽

Fura 2 ◽

Synergistic Inhibitory Effect ◽

Dose Dependent

SummaryProbenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.

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In vivo anti-tumor efficacy of Croton oblongifolius in DMBA induced mammary tumor in rats

Indian Journal of Animal Research ◽

10.18805/ijar.b-3301 ◽

2017 ◽

Author(s):

Gowrav Adiga P. ◽

N. B. Shridhar ◽

Jagadeesh S. Sanganal ◽

Suguna Rao ◽

N. Shilpa ◽

...

Keyword(s):

Mammary Tumor ◽

Stem Bark ◽

Dependent Manner ◽

Sprague Dawley ◽

Treatment Groups ◽

Sprague Dawley Rats ◽

Croton Oblongifolius ◽

Anti Tumor Activity ◽

Dose Dependent

The present study was designed to evaluate anti-tumor activity of the methanolic extract of stem bark of Croton oblongifolius in Sprague Dawley rats. The tumor was induced in rats by DMBA given orally and intramammarily and challenging with plant extract. After obtaining suitable mass of tumor, the extract was gavaged to rats @ 200, 500 and 800 mg/kg which showed reduction in mammary tumor volume in dose-dependent manner, which was supported by histopathological observations of the treatment groups.

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Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration

AJP Renal Physiology ◽

10.1152/ajprenal.1999.276.5.f700 ◽

1999 ◽

Vol 276 (5) ◽

pp. F700-F710 ◽

Cited By ~ 7

Author(s):

Max Salomonsson ◽

William J. Arendshorst

Keyword(s):

Blood Flow ◽

Dependent Manner ◽

Free Solution ◽

Sprague Dawley ◽

Vascular Effects ◽

Renal Vasculature ◽

Afferent Arterioles ◽

Renal Vascular ◽

Dose Dependent

This study provides new information about the relative importance of Ca2+ mobilization and entry in the renal vascular response to adrenoceptor activation. We measured renal blood flow (RBF) in Sprague-Dawley rats in vivo using electromagnetic flowmetry. We measured intracellular free Ca2+ concentration ([Ca2+]i) in isolated afferent arterioles utilizing ratiometric photometry of fura-2 fluorescence. Renal arterial injection of NE produced a transient decrease in RBF. The response was attenuated, in a dose-dependent manner, up to ∼50% by nifedipine, an antagonist of L-type Ca2+ entry channels. Inhibition of Ca2+ mobilization by 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8) inhibited the renal vascular effects of NE in a dose-dependent manner, with maximal blockade of ∼80%. No additional attenuation was observed when nifedipine and TMB-8 were administered together. In microdissected afferent arterioles, norepinephrine (NE; 10−6 M) elicited an immediate square-shaped increase in [Ca2+]i, from 110 to 240 nM. This in vitro response was blocked by nifedipine (10−6 M) and TMB-8 (10−5 M) to a degree similar to that of the in vivo experiments. A nominally calcium-free solution blocked 80–90% of the [Ca2+]iresponse to NE. The increased [Ca2+]ielicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine. In contrast, TMB-8 had no effect. Our results indicate that both Ca2+ entry and mobilization play important roles in the renal vascular Ca2+ and contractile response to adrenoceptor activation. The entry and mobilization mechanisms activated by NE may interact. That a calcium-free solution caused a larger inhibition of the NE effects on afferent arterioles than nifedipine suggests more than one Ca2+ entry pathway.

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Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650532 ◽

1996 ◽

Vol 76 (01) ◽

pp. 111-117 ◽

Cited By ~ 10

Author(s):

Yasuto Sasaki ◽

Junji Seki ◽

John C Giddings ◽

Junichiro Yamamoto

Keyword(s):

Blood Flow ◽

Thrombus Formation ◽

Dependent Manner ◽

Red Cell ◽

Cell Velocity ◽

Red Cell Velocity ◽

The Mean ◽

Wall Shear ◽

Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

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Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

Cell Death and Disease ◽

10.1038/s41419-021-03701-z ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Xuxing Shen ◽

Chao Wu ◽

Meng Lei ◽

Qing Yan ◽

Haoyang Zhang ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitor ◽

Osteoclast Differentiation ◽

Dependent Manner ◽

Serum Enzyme ◽

Herg Channels ◽

Anti Tumor Activity ◽

Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

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Effect of Synchronous Versus Sequential Regimens on the Pharmacokinetics and Biodistribution of Regorafenib with Irradiation

Pharmaceutics ◽

10.3390/pharmaceutics13030386 ◽

2021 ◽

Vol 13 (3) ◽

pp. 386

Author(s):

Tung-Hu Tsai ◽

Yu-Jen Chen ◽

Li-Ying Wang ◽

Chen-Hsi Hsieh

Keyword(s):

Stereotactic Body Radiation Therapy ◽

In Vivo Studies ◽

Control Group ◽

High Dose ◽

Dependent Manner ◽

Hep G2 ◽

Time Curve ◽

Dose Dependent

This study was performed to evaluate the interaction between conventional or high-dose radiotherapy (RT) and the pharmacokinetics (PK) of regorafenib in concurrent or sequential regimens for the treatment of hepatocellular carcinoma. Concurrent and sequential in vitro and in vivo studies of irradiation and regorafenib were designed. The interactions of RT and regorafenib in vitro were examined in the human hepatoma Huh-7, HA22T and Hep G2 cell lines. The RT–PK phenomenon and biodistribution of regorafenib under RT were confirmed in a free-moving rat model. Regorafenib inhibited the viability of Huh-7 cells in a dose-dependent manner. Apoptosis in Huh-7 cells was enhanced by RT followed by regorafenib treatment. In the concurrent regimen, RT decreased the area under the concentration versus time curve (AUC)regorafenib by 74% (p = 0.001) in the RT2 Gy × 3 fraction (f’x) group and by 69% (p = 0.001) in the RT9 Gy × 3 f’x group. The AUCregorafenib was increased by 182.8% (p = 0.011) in the sequential RT2Gy × 1 f’x group and by 213.2% (p = 0.016) in the sequential RT9Gy × 1 f’x group. Both concurrent regimens, RT2Gy × 3 f’x and RT9Gy × 3 f’x, clearly decreased the biodistribution of regorafenib in the heart, liver, lung, spleen and kidneys, compared to the control (regorafenib × 3 d) group. The concurrent regimens, both RT2Gy × 3 f’x and RT9Gy × 3 f’x, significantly decreased the biodistribution of regorafenib, compared with the control group. The PK of regorafenib can be modulated both by off-target irradiation and stereotactic body radiation therapy (SBRT).

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Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host–endophyte interaction in vitro and in vivo

Canadian Journal of Microbiology ◽

10.1139/w06-141 ◽

2007 ◽

Vol 53 (3) ◽

pp. 380-390 ◽

Cited By ~ 36

Author(s):

Pious Thomas ◽

Sima Kumari ◽

Ganiga K. Swarna ◽

T.K.S. Gowda

Keyword(s):

Culture Medium ◽

Carica Papaya ◽

In Vitro Cultures ◽

Dependent Manner ◽

16S Rdna Sequence ◽

16S Rdna Sequence Analysis ◽

Single Organism ◽

Dose Dependent

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (~1 cm) of papaya (Carica papaya L. ‘Surya’) planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2–4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea ( P. ananatis ), Enterobacter ( E. cloacae ), Brevundimonas ( B. aurantiaca ), Sphingomonas , Methylobacterium ( M. rhodesianum ), and Agrobacterium ( A. tumefaciens ) or two Gram-positive genera, Microbacterium ( M. esteraromaticum ) and Bacillus ( B. benzoevorans ) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550 = 0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.

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