Biochemical regulation of breast cancer cell expression of S1P2 (Edg-5) and S1P3 (Edg-3) G protein-coupled receptors for sphingosine 1-phosphate

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.     

Bacterial glycoproteins: functions,biosynthesis and applications

Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms. The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time. Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms. Most studied prokaryotic glycoproteins are the S-layer glycoproteins of Archeabacteria. Apart from these, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins. In general, prokaryotes are deprived of the cellular organelles required for glycosylation. In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes. Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce. This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated. Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon.     

Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum

The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 microM, which is a ratio of 1:40,000 hemes, can elevate 10-fold in some of the anemias associated with malaria disease protection. This work examines a binding mechanism for zinc protoporphyrin IX inhibition of heme crystallization similar to the antimalarial quinolines. Zinc protoporphyrin IX neither forms crystals alone nor extends on preformed heme crystals. Inhibition of both seed heme crystal formation and crystal extension occurs with an inhibitory concentration (IC)50 of 5 microM. Field emission in-lens scanning electron microscopy depicts the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of preformed hemozoin templates. In vitro zinc protoporphyrin IX, like the quinolines, binds to heme crystals in a saturable, specific, pH, and time-dependent manner. The ratio at saturation is approximately 1 zinc protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc protoporphyrin IX to heme ratio 6 to 10 times that in the erythrocyte cytosol, which also corresponds to elevated ratios found in heme crystals purified from Plasmodium parasites. This work implicates protection from malaria by a mechanism where elevated zinc protoporphyrin IX in anemic erythrocytes binds to heme crystals to inhibit further crystallization. In endemic malaria areas, severe iron deficiency anemia should be treated with antimalarials along with iron replenishment.     

Polyunsaturated fatty acids (PUFA) regulate neurotransmitter contents in rat brain

The effects of feeding of 6-propylthiouracil (6-PTU) and polyunsaturated fatty acids (PUFA) independently and in combination and administration (ip) of a single dose of triiodothyronine (T3) (2.5 microg/100 g body wt) along with feeding of 6-PTU and PUFA were studied in rat brain. Dopamine (DA), 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxy indole acetic acid (5-HIAA), norepinephrine (NE) and epinephrine (EPI) contents were assayed in the hypothalamus and cerebral cortex regions. It was found that 6-PTU feeding resulted in decrease in dopamine, 5-HT, 5-HTP and 5-HIAA in both regions. In animals fed with PUFA followed by administration of T3, the DA level was found normal.     

Male reproductive toxicity of vincristine: ultrastructural changes in the epididymal epithelial apical cell

The toxic effect of vincristine on the apical cells of the rat caput epididymis was investigated. The drug was administered at 20 and 40 microg/kg body weight daily for 15 days. Light microscopy using semithin sections, and transmission electron microscopy, of the caput epididymis were undertaken. The results revealed that the basal region of the apical cell was in contact with the basement membrane and the luminal end took part in endocytosis. The apical cells reflected a dose-dependent response to vincristine (VCR) treatment. In general the changes included protrusion of the apical ends deep into the lumen, with the nucleus of the cell located in such protruded ends, and an increase in the abundance of lysosomal bodies and multivesicular bodies. These changes reflected the physiological response of the apical cell to VCR treatment rather than toxic manifestations.     

Regulation of Adherence and Virulence by the Entamoeba histolytica Lectin Cytoplasmic Domain, Which Contains a β2 Integrin Motif

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the β2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin’s role in virulence.     

Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01196871","term_id":"NCT01196871"}}NCT01196871     

Impact of an Interdisciplinary Deep Brain Stimulation Screening Model on Post-Surgical Complications in Essential Tremor Patients

Objective

To investigate the relationship of our interdisciplinary screening process on post-operative unintended hospitalizations and quality of life.

Background

There are currently no standardized criteria for selection of appropriate Deep Brain Stimulation candidates and little hard data exists to support the use of any singular method.

Methods

An Essential Tremor cohort was selected from our institutional Deep Brain Stimulation database. The interdisciplinary model utilized seven specialties who pre-operatively screened all potential Deep Brain Stimulation candidates. Concerns for surgery raised by each specialty were documented and classified as none, minor, or major. Charts were reviewed to identify unintended hospitalizations and quality of life measurements at 1 year post-surgery.

Results

Eighty-six percent (44/51) of the potential screened candidates were approved for Deep Brain Stimulation. Eight (18%) patients had an unintended hospitalization during the follow-up period. Patients with minor or major concerns raised by any specialty service had significantly more unintended hospitalizations when compared to patients without concerns (75% vs. 25%, p < 0.005). The rate of hospitalization revealed a direct relationship to the “level of concern”; ranging from 100% if major concerns, 42% if minor concerns, and 7% if no concerns raised, p = 0.001. Quality of life scores significantly worsened in patients with unintended hospitalizations at 6 (p = 0.046) and 12 months (p = 0.027) when compared to baseline scores. No significant differences in tremor scores between unintended and non-unintended hospitalizations were observed.

Conclusions

The number and level of concerns raised during interdisciplinary Deep Brain Stimulation screenings were significantly related to unintended hospitalizations and to a reduced quality of life. The interdisciplinary evaluation may help to stratify risk for these complications. However, data should be interpreted with caution due to the limitations of our study. Further prospective comparative and larger studies are required to confirm our results.     

The Role of ARF6 in Biliary Atresia

Background & Aims

Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods

To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results

Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10^-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10^-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10^-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10^-7), ERK/MAPK and CREB canonical pathways (p<1 x10^-34), and functional networks for cellular development and proliferation (p<1 x10^-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions

The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.