Syndecan-4 promotes the retention of phosphatidylinositol 4,5-bisphosphate in the plasma membrane

Although phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates syndecan-4 function, the potential influence of syndecan-4 on PIP₂ remains unknown. GFP containing PIP₂-binding-PH domain of phospholipase Cδ (GFP-PHδ) was used to monitor PIP₂. Syndecan-4 overexpression in COS-7 cells enhanced membrane translocation of GFP-PHδ, while the opposite was observed when syndecan-4 was knocked-down. PIP₂ levels were higher in total phospholipids extracted from rat embryo fibroblasts expressing syndecan-4. Syndecan-4-induced membrane targeting of GFP-PHδ was further enhanced by phosphoinositide-3-kinase inhibitor, but not by phospholipase C (PLC) inhibitor. Besides, both ionomycin and epidermal growth factor caused dissociation of GFP-PHδ from plasma membrane, an effect that was significantly delayed by syndecan-4 over-expression. Collectively, these data suggest that syndecan-4 promotes plasma membrane retention of PIP₂ by negatively regulating PLC-dependent PIP₂ degradation.     

An iron(II) complex with a N3S2 thioether ligand in the generation of an iron(IV)-oxo complex and its reactivity in olefin epoxidation

A new iron(II) complex bearing an N3S2 thioether ligand was synthesized and characterized with various spectroscopic techniques and X-ray crystallography. A mononuclear nonheme iron(IV)-oxo intermediate was generated in the reaction of the iron(II) complex with various terminal oxidants. The iron(IV)-oxo species show a capability of epoxidizing olefins, and the olefin epoxidation occurs via an electrophilic oxidation mechanism.     

Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.     

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Synthesis and Biological Evaluation of 9-Deazaadenine 5′-Deoxy-6′,6′-Difluoro-Carbocyclic C-Nucleoside Phosphonic Acid Derivatives

The first synthetic route to 5′-deoxy-6′,6′-difluoro-carbocyclic C-nucleoside [9-deazaadenosine, (pyrrolo[3,2-d]pyrimidine)] phosphonic acids from commercially available epichlorohydrin 5 was described. The key C-C bond formation from cyclopentanone to base precursor was performed using the Knoevenagel-type condensation. Synthesized C-nucleoside phosphonic acids were tested for anti-HIV and anti-leukemic activities. They showed moderate cytotoxicity-derived anti-HIV and anti-leukemic activities.     

Muscle mass,structural and functional investigations of senescence-accelerated mouse P8 (SAMP8)

Sarcopenia is an age-related systemic syndrome with progressive deterioration in skeletal muscle functions and loss in mass. Although the senescence-accelerated mouse P8 (SAMP8) was reported valid for muscular ageing research, there was no report on the details such as sarcopenia onset time. Therefore, this study was to investigate the change of muscle mass, structure and functions during the development of sarcopenia. Besides the average life span, muscle mass, structural and functional measurements were also studied. Male SAMP8 animals were examined at month 6, 7, 8, 9, and 10, in which the right gastrocnemius was isolated and tested for ex vivo contractile properties and fatigability while the contralateral one was harvested for muscle fiber cross-sectional area (FCSA) and typing assessments. Results showed that the peak of muscle mass appeared at month 7 and the onset of contractility decline was observed from month 8. Compared with month 8, most of the functional parameters at month 10 decreased significantly. Structurally, muscle fiber type IIA made up the largest proportion of the gastrocnemius, and the fiber size was found to peak at month 8. Based on the altered muscle mass, structural and functional outcomes, it was concluded that the onset of sarcopenia in SAMP8 animals was at month 8. SAMP8 animals at month 8 should be at pre-sarcopenia stage while month 10 at sarcopenia stage. It is confirmed that SAMP8 mouse can be used in sarcopenia research with established time line in this study.     

Dopamine D3 Receptor Mediates Preadolescent Stress-Induced Adult Psychiatric Disorders

Several studies have shown that repeated stressful experiences during childhood increases the likelihood of developing depression- and anxiety-related disorders in adulthood; however, the underlying mechanisms are not well understood. We subjected drd3-EGFP and drd3-null mice to daily, two hour restraint stress episodes over a five day period during preadolescence (postnatal day 35 to 39), followed by social isolation. When these mice reached adulthood (post-natal day > 90), we assessed locomotor behavior in a novel environment, and assessed depression-related behavior in the Porsolt Forced Swim test. We also measured the expression and function of dopamine D3 receptor in limbic brain areas such as hippocampus, nucleus accumbens and amygdala in control and stressed drd3-EGFP mice in adulthood. Adult male mice subjected to restraint stress during preadolescence exhibited both anxiety- and depression-related behaviors; however, adult female mice subjected to preadolescent restraint stress exhibited only depression-related behaviors. The development of preadolescent stress-derived psychiatric disorders was blocked by D3 receptor selective antagonist, SB 277011-A, and absent in D3 receptor null mice. Adult male mice that experienced stress during preadolescence exhibited a loss of D3 receptor expression and function in the amygdala but not in hippocampus or nucleus accumbens. In contrast, adult female mice that experienced preadolescent stress exhibited increased D3 receptor expression in the nucleus accumbens but not in amygdala or hippocampus. Our results suggest that the dopamine D3 receptor is centrally involved in the etiology of adult anxiety- and depression-related behaviors that arise from repeated stressful experiences during childhood.     

Mapping and Congenic Dissection of Genetic Loci Contributing to Hyperglycemia and Dyslipidemia in Mice

Background

Patients with dyslipidemia have an increased risk of developing type 2 diabetes, and diabetic patients often have dyslipidemia. Potential genetic connections of fasting plasma glucose with plasma lipid profile were evaluated using hyperlipidemic mice.

Methods

225 male F₂ mice were generated from BALB/cJ (BALB) and SM/J(SM) Apoe-deficient (Apoe^−/−) mice and fed a Western diet for 5 weeks. Fasting plasma glucose and lipid levels of F₂ mice were measured before and after 5 weeks of Western diet and quantitative trait locus (QTL) analysis was performed using data collected from these two time points. 144 SNP(single nucleotide polymorphism) markers across the entire genome were typed.

Results

One major QTL (logarithm of odds ratio (LOD): 6.46) peaked at 12.7 cM on chromosome 9,Bglu16, and 3 suggestive QTLs on chromosomes 15, 18 and X were identified for fasting glucose, and over 10 loci identified for lipid traits. Bglu16 was adjacent to a major QTL, Hdlq17, for high-density lipoprotein (HDL) cholesterol (LOD: 6.31, peak: 19.1 cM). A congenic strain with a donor chromosomal region harboring Bglu16 and Hdlq17 on the Apoe^−/− background showed elevations in plasma glucose and HDL levels. Fasting glucose levels were significantly correlated with non-HDL cholesterol and triglyceride levels, especially on the Western diet, but only marginally correlated with HDL levels in F₂ mice.

Conclusions

We have demonstrated a correlative relationship between fasting glucose and plasma lipids in a segregating F₂ population under hyperlipidemic conditions, and this correlation is partially due to genetic linkage between the two disorders.     

Clinical Assessment of Lamina Cribrosa Curvature in Eyes with Primary Open-Angle Glaucoma

Purpose

Quantitative evaluation of lamina cribrosa (LC) posterior bowing in primary open-angle glaucoma (POAG) eyes using swept-source optical coherence tomography.

Methods

Patients with POAG (n = 123 eyes) and healthy individuals of a similar age (n = 92 eyes) were prospectively recruited. Anterior laminar insertion depth (ALID) was defined as the vertical distance between the anterior laminar insertion and a reference plane connecting the Bruch’s membrane openings (BMO). The mean LC depth (mLCD) was approximated by dividing the area enclosed by the anterior LC, the BMO reference plane, and the two vertical lines for ALID measurement by the length between those two vertical lines. The LC curvature index was defined as the difference between the mLCD and the ALID. The factors influencing the LC curvature index were evaluated.

Results

The ALID and mLCD were significantly larger in POAG eyes than in healthy controls (P < 0.05). The LC curvature index was significantly larger in POAG eyes than in healthy controls on both the horizontal (85.8 ± 34.1 vs. 68.2 ± 32.3 μm) and vertical meridians (49.8 ± 38.5 vs. 32.2 ± 31.1 μm, all P < 0.001). Multivariate regression showed significant associations of greater disc area (P < 0.001), vertical C/D ratio (P < 0.001) and mLCD (P < 0.001), smaller rim area (P = 0.001), thinner average RNFLT (P < 0.001), and myopic refraction (P = 0.049) with increased LC curvature index. There was no difference in the LC curvature index between mild (MD > –6 dB) and moderate-to-advanced glaucoma (MD < –6 dB, P = 0.95).

Conclusions

LC posterior bowing was increased in POAG eyes, and was significantly associated with structural optic nerve head (ONH) changes but not with functional glaucoma severity. Quantitative evaluation of LC curvature can facilitate assessment of glaucomatous ONH change.