Spatial and temporal regulation of tenascin-R glycosylation in the cerebellum

The cellular adhesion molecule tenascin-R is a multifunctional extracellular matrix component expressed exclusively in the central nervous system. The expression of tenascin-R by oligodendrocytes and small interneurons in the hippocampus and cerebellum is highly regulated during development of these regions. This complex glycoprotein displays both adhesive and anti-adhesive properties that contribute to the formation and maintenance of synapses. We have determined that tenascin-R associated with Purkinje cell bodies and their dendrites in the molecular layer of the cerebellum bears N-linked oligosaccharides terminating with beta1,4-linked GalNAc-4-SO(4), whereas tenascin-R in other regions of the cerebellum does not bear this modification. Expression of this unique sulfated carbohydrate structure is also temporally regulated, increasing throughout cerebellar development. The most dramatic increase in GalNAc-4-SO(4) occurs between postnatal days 14 and 21, corresponding to a period of Purkinje cell dendrite extension and synaptogenesis. The spatially and temporally regulated addition of this unique sulfated carbohydrate to tenascin-R may serve to modulate its adhesive/anti-adhesive or other biological properties in vivo.     

Characterization of proteins in flagellates and growing amebae of Naegleria fowleri.

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.     

Power Estimation for Two-Sample Tests Using Importance and Antithetic Resampling

Collings and Hamilton (1988), described a uniform bootstrap method that is applied on observed or pilot data in order to approximate the power of the two-sample Wilcoxon test for location shift alternatives. In this paper we demonstrate how importance and antithetic resampling can be used to substantially reduce the amount of computation needed to approximate the power of the two-sample tests for location shift and scale alternatives. Importance and antithetic bootstrap resampling methods are applied to simulated data of different sample sizes from a variety of distributions as well as to data from the Iowa 65+ Rural Health Study. Also, a suggestion is given for using a combination of importance and antithetic resampling for approximating the power of two-sample tests.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

Identification of platination sites on human serum transferrin using 13C and 15N NMR spectroscopy

 Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl₂] or cis-[PtCl₂(NH₃)₂] have been investigated in solution via observation of [¹H,¹⁵N] NMR resonances of the Pt complexes, [¹H,¹³C] resonances of the eCH₃ groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days. Received: 5 May 1999 / Accepted: 26 July 1999     

Insulin resistance and decreased spexin in Indian Patients with Type 2 Diabetes Mellitus

Spexin is novel biomarker, which plays a potential role in glucose and lipid metabolisms. However, there was paucity of serum spexin levels in obesity and diabetes mellitus subjects. Hence the current study was aimed to find the relationship between the serum spexin levels in type 2 Diabetes mellitus (type 2 DM) with extrapolation of cardiovascular disease (CVD) risk. A cross-sectional study included 330 participants, subdivided as control (n=110), type 2 DM (n=110) and type 2 DM with CVD groups (n=110). HbA1c, insulin, lipid profile, spexin & leptin including blood pressure and body mass index were analyzed from all the participants. The serum spexin levels (ng/ml) were significantly decreased in type 2 DM (mean ± sd: 0.65 ± 0.03) and type 2 DM with CVD (0.48 ± 0.02) groups compared to the control (0.79 ± 0.03) group (p<0.001). The decreased spexin levels were observed in type 2 DM, and further more decreased in type 2 DM with CVD patients compared to controls indicating that spexin levels could be served as an early prediction of obesity-induced T2DM with CVD risk.     

In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection

Although dendritic cells (DC) are potent APC that prime T cells against many pathogens, there is no direct evidence that DC are required for immunity to Mycobacterium tuberculosis (Mtb) infection. The requirement for DC to prime the CD4+ T cell response following Mtb infection was investigated using pCD11c-diptheria toxin receptor/GFP transgenic mice, in which DC can be transiently ablated in vivo. We show a critical role for DC in initiation of the CD4+ T cell response to the mycobacterial Ag early secretory Ag of tuberculosis 6. The delay in initiating the Ag-specific T cell response led to impaired control of Mtb replication. Interestingly, DC were not required for the secondary CD4+ T cell response following Mtb infection in peptide-vaccinated mice. Thus, this study shows that DC are essential for the initiation of the adaptive T cell response to the human pathogen Mtb.     

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.