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Haitao Hu

Publications and source records attributed to Haitao Hu.

16 recordsLinked to original sources
PubMed2006

Vaccine-delivered HIV envelope inhibits CD4(+) T-cell activation, a mechanism for poor HIV vaccine responses.

The human immunodeficiency virus (HIV) causes impairment of the immune system in part by targeting CD4(+) T cells for infection and dysfunction. HIV envelope (Env) present on free virions and infected cells causes dysfunction of uninfected bystander CD4(+) T cells via interaction with both CD4 and coreceptors. Env is commonly used as part of a cocktail of HIV antigens in current vaccines. In DNA and viral vector vaccine approaches, antigen-presenting cells (APCs) and non-APCs in the vicinity of the vaccine delivery site and draining lymph node express vaccine-derived antigens. The studies here demonstrate that cell-surface expression of Env on APCs and non-APCs as part of the vaccine action causes an inhibition of antigen-induced CD4(+) T-cell activation and proliferation mediated by CD4 binding and suggests a potential mechanism for reduced activity of Env-containing HIV vaccines. Similar studies using a functional Env lacking CD4 binding circumvented suppression, suggesting an alternative and potentially superior approach to HIV vaccine design.

AIDS Vaccines↗
PubMed2006

Revisiting the initial rate approximation in kinetic NOE measurements.

The nuclear Overhauser effect (NOE) is undoubtedly one of the most useful tools in NMR spectroscopy and is widely used in solving structural and conformational problems of small organic molecules and macromolecular systems alike. In particular, measurement of the kinetics of the NOE, often facilitated by selective 1D NOE buildup experiments, can generate invaluable quantitative distance information for the molecule being investigated. In practice, analysis of such kinetic NOE data routinely assumes a first-order approximation of the initial buildup rate. However, often times such an approximation holds true only for the shortest mixing times. As shown by Macura and others, the linear range of the NOE buildup obtained from 2D NOESY and exchange experiments can be substantially extended by simply scaling the NOE cross-peaks against the corresponding diagonal peaks. In this note, we demonstrate through a detailed analysis that the same approach can be applied to the analysis of 1D NOE data obtained with the DPFGSE NOE pulse sequence, one of the most widely used selective 1D NOE experiments today. We show that this approach allows the inclusion of data points acquired with much longer mixing times in the analysis and thus considerably improves the accuracy of the measured cross-relaxation rates and internuclear distances, while considerably simplifying the data analysis. Similar results can be obtained for the rotating frame DPFGSE ROE experiment.

Journal Article↗
PubMed2006

The biochemical effect of Ser167 phosphorylation on Chlamydomonas reinhardtii centrin.

Centrin is an EF-hand calcium-binding protein found in microtubule organizing centers of organisms ranging from algae and yeast to man. Phosphorylation in the centrin C-terminal domain occurs in mitosis and is associated with alterations in contractile fibers. To obtain insight into the structural basis for the functional effect of phosphorylation, Chlamydomonas reinhardtii centrin C-terminal domain phosphorylated at Ser167 (pCRC-C) has been produced and characterized. The structure of pCRC-C was compared to the unmodified protein by NMR spectroscopy. The effect of phosphorylation on target binding was examined for the complex of pCRC-C and a 19 residue centrin-binding fragment of Kar1. Remarkably, the efficient and selective phosphorylation by PKA was suppressed in the complex. Moreover, comparisons of NMR chemical shift differences induced by phosphorylation reveal a greater effect from phosphorylation in the context of the Kar1 complex than for the free protein. These results directly demonstrate that phosphorylation modulates the structure and biochemical activities of centrin.

Animals↗
PubMed2005

Structure of the N-terminal calcium sensor domain of centrin reveals the biochemical basis for domain-specific function.

Centrin is an essential component of microtubule-organizing centers in organisms ranging from algae and yeast to humans. It is an EF-hand calcium-binding protein with homology to calmodulin but distinct calcium binding properties. In a previously proposed model, the C-terminal domain of centrin serves as a constitutive anchor to target proteins, and the N-terminal domain serves as the sensor of calcium signals. The three-dimensional structure of the N-terminal domain of Chlamydomonas rheinhardtii centrin has been determined in the presence of calcium by solution NMR spectroscopy. The domain is found to occupy an open conformation typical of EF-hand calcium sensors. Comparison of the N- and C-terminal domains of centrin reveals a structural and biochemical basis for the domain specificity of interactions with its cellular targets and the distinct nature of centrin relative to other EF-hand proteins. An NMR titration of the centrin N-terminal domain with a fragment of the known centrin target Sfi1 reveals binding of the peptide to a discrete site on the protein, which supports the proposal that the N-terminal domain serves as a calcium sensor in centrin.

Animals↗
PubMed2005

Dynamics of hydrogen-deuterium exchange in Chlamydomonas centrin.

Chlamydomonas reinhardtii centrin is a 169-amino acid residue calcium binding protein belonging to the EF-hand protein superfamily. Centrin is associated with the microtubule organizing center (MTOC) in all eukaryotes, and in Chlamydomonas, centrin is a component of the flagellar basal body apparatus. Recombinant full-length centrin, calmodulin, and terminal domain fragments [Ccen-N (residues 1-94) and Ccen-C (residues 99-169)] were used to examine hydrogen-deuterium (H --> D) exchange dynamics using combined attenuated total reflectance (ATR) Fourier transform-infrared (FT-IR) spectroscopy, curve fit, and two-dimensional correlation analysis. Analysis of the Ccen-N and Ccen-C fragments allowed separation of domain specific solvent exchange events and together with analysis of the full-length proteins provides novel insight into domain accessibility to the aqueous environment and the internal dynamics of the protein.

Amino Acid Sequence↗
PubMed2005

Simplifying DOSY spectra with selective TOCSY edited preparation.

Diffusion-ordered NMR spectroscopy, while quite powerful, is limited by its inability to resolve signals that are severely overlapped in the proton spectrum. We present here a DOSY experiment that uses selective TOCSY as an editing/preparation period. With this method, well-resolved signals of the analytes are selectively excited and the magnetization subsequently transferred by isotropic mixing to resonances buried in the matrix background, which are then resolved by the ensuing DOSY sequence. Key to the success of our proposed method is the incorporation of a highly effective zero-quantum filter into the selective TOCSY preparation period, which prevents zero-quantum coherence from being carried into the DOSY part of the pulse sequence. Further improvement in spectral resolution can be obtained by expanding the proposed experiment into a 3D sequence and utilizing the homonuclear decoupling feature of the BASHD-TOCSY technique. Both pulse sequences were found to greatly simplify the DOSY spectrum of a 'dirty' sucrose/raffinose mixture, as the complex matrix background is no longer present to obscure or overlap with the signals of interests. Furthermore, complete resolution of the relevant signals was achieved with the 3D sequence.

Carbohydrates↗
PubMed2005

A band-selective composite gradient: application to DQF-COSY.

We describe a unique band-selective method that utilizes a selective composite gradient to simultaneously achieve band selection and coherence pathway selection. This element is similar to the composite gradient known as the CLUB sandwich except the original broadband pulses have been replaced with selective pulses and the strengths of the antipolar gradients have been unbalanced. In this way, only the signals within the inversion band will continue to dephase throughout the duration of the element and satisfy the proper encoding-to-decoding gradient ratio necessary for coherence selection. Apart from the inverted polarity and asymmetry of the gradients, the band-selective CLUB sandwich is identical to the DPFGSE sequence and provides many of its desirable characteristics. We have successfully incorporated the band-selective CLUB into the DQF-COSY pulse sequence to create a band-selective experiment that offers the selectivity desired for resolution enhancement while maintaining excellent phase behavior. This is demonstrated on the congested aliphatic region of the ionophorous antibiotic Lasalocid A.

Algorithms↗
PubMed2005

[Learning and memory amelioration of transplantation of the neural stem cells modified with human brain-derived neurotrophic factor gene on Alzheimer disease model rat].

OBJECTIVE: To investigate the memory amelioration of the Alzheimer disease (AD) model rat after being transplanted the single neural stem cells (NSC) and NSC modified with human brain-derived neurotrophic factor (hBDNF) gene. METHODS: Forty SD rats were divided evenly into 4 groups randomly. The AD model rats were made by cutting unilaterally the fibria-fornix of male rats. Ten to twelve days after surgery, the genetically modified and unmodified NSC were implanted into the lateral cerebral ventricle of group III and group IV respectively. Two weeks after transplantation, the amelioration of memory impairment of the rats was detected by Morris water maze. RESULTS: The average escaping latency of the group III and group IV (41.84 +/- 21.76 s, 25.23 +/- 17.06 s respectively) was shorter than that of the group II (70.91 +/- 23.67 s) (P<0.01). The percentage of swimming distance in the platform quadrant in group III (36.9%) and in group IV (42.0%) was higher than that in the group II (26.0%) (P<0.01). More marginal and random strategies were used in group II. The percentage of swimming distance in the platform quadrant in group IV was also greater than that in group III (P<0.05). There were no significant differences in the average escaping latency, the percentage of swimming distance in the platform quadrant and the probe strategy between group IV and group I (P>0.05). More lineal and oriented strategies were used in group IV. CONCLUSION: The behavioral amelioration of AD model rat was obtained by transplanting single NSC and hBDNF-gene-modified NSC. The effect of the NSC group modified with hBDNF gene is better than that of the group III.

Alzheimer Disease↗
PubMed2005

[The optimization of the method of culturing neural stem cells in neonatal rat brain].

OBJECTIVE: To establish a better method of isolating and culturing of neural stem cells (NSCs) in neonatal rat brain. METHODS: Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used to culture NSCs of neonatal rat. The culture concentration used were 1 x 10(4), 1 x 10(5), 1 x 10(6), and 1 x 10(7)/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate as to be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. RESULTS: The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining long-term survival in vitro. The cells could be differentiated into neurons and glia. It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS) into the medium at the beginning of the culturing. When 10% FBS was added into the medium, the neurospheres differentiated quickly. When concentration 1 x 10(6)/ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. CONCLUSION: A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.

Actihaemyl↗
PubMed2005

[The effects of recombinant human erythropoietin on electroretinogram after raising intraocular pressure in rabbits].

PURPOSE: To investigate the protective effect of recombinant human erythropoietin (rhEPO) on retinal ischemic injury induced by raising intraocular pressure. METHODS: Twelve New Zealand rabbits were divided into the model group (n=6) and the EPO group (n=6). Either of bilateral eyes was randomly made into the acutely high intraocular pressure model using the method of saline perfusion into anterior chamber. After the model was made, rhEPO 100 IU x kg(-1) was injected into the hypodermic tissue of EPO group rabbits twice a week for one week. Flash electroretinogram (ERG) was detected 30 min before and at 1, 3, 7, and 14 days after perfusion, respectively. RESULTS: The amplitude of ERG-b wave had no significant difference in the EPO group compared with that in the model group before anterior chamber perfusion (P > 0.05). The amplitude of ERG-b wave dropped down to the lowest at 1 day after perfusion and could not come back to the baseline in the model group (P < 0.05). The resemble situation was in the EPO group except that the amplitude of ERG-b wave came back to the baseline at 14 days (P > 0.05). CONCLUSION: EPO can improve the amplitude of ERG-b wave following retinal ischemia. This indicates that EPO has the potential to be an optimal neuroprotective agent.

Animals↗
PubMed2004

The mode of action of centrin. Binding of Ca2+ and a peptide fragment of Kar1p to the C-terminal domain.

Centrin is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. It is found in microtubule-organizing centers of organisms ranging from algae and yeast to man. In vitro, the C-terminal domain of centrin binds to the yeast centrosomal protein Kar1p in a calcium-dependent manner, whereas the N-terminal domain does not show any appreciable affinity for Kar1p. To obtain deeper insights into the structural basis for centrin's function, we have characterized the affinities of the C-terminal domain of Chlamydomonas reinhardtii centrin for calcium and for a peptide fragment of Kar1p using CD, fluorescence, and NMR spectroscopy. Calcium binding site IV in C. reinhardtii centrin was found to bind Ca2+ approximately 100-fold more strongly than site III. In the absence of Ca2+, the protein occupies a mixture of closed conformations. Binding of a single ion in site IV is sufficient to radically alter the conformational equilibrium, promoting occupancy of an open conformation. However, an exchange between closed and open conformations remains even at saturating levels of Ca2+. The population of the open conformation is substantially stabilized by the presence of the target peptide Kar1p-(239-257) to a point where a single ion bound in site IV is sufficient to completely shift the conformational equilibrium to the open conformation. This is reflected in the enhancement of the Ca2+ affinity in this site by more than an order of magnitude. These data confirm the direct coupling of the Ca2+ binding-induced shift in the equilibrium between the closed and open conformations to the binding of the peptide. Combined with the common localization of the two proteins in the microtubule organizing center, our results suggest that centrin is constitutively bound to Kar1p through its C-terminal domain and that centrin's calcium sensor activities are mediated by the N-terminal domain.

Animals↗
PubMed2004

Extending the limits of the selective 1D NOESY experiment with an improved selective TOCSY edited preparation function.

Compared to its 2D counterpart, the selective 1D NOESY experiment offers greatly simplified spectral interpretation and is invaluable to the structure elucidation of small-to-medium sized molecules, although its application is limited to well-resolved resonances only. The doubly selective 1D TOCSY-NOESY experiment allows the 1D NOESY experiment to be extended to resonances within overlapped spectral regions. However, existing methods do not address the critical issue of zero-quantum interference, which leads to severe anti-phase distortions to the line shape of scalar coupled spins and often complicates the identification of weak NOE enhancements. In this communication, we describe an improved selective TOCSY edited preparation (STEP) function and its application to the selective 1D NOESY experiment. The STEP function incorporates a novel zero-quantum filter introduced by Thrippleton and Keeler [Angew. Chem. Int. Ed. 42 (2003) 3938], which permits essentially complete suppression of zero-quantum coherence in a single scan. Residual anti-phase distortions due to spin-state mixing are removed using the double difference methodology reported by Shaka et al. [45th Experimental NMR Conference, Pacific Grove, USA, 2004]. The combined use of these techniques ensures that the final spectra are free of distortions, which is crucial to the reliable detection of weak NOE enhancements. Although employed as an additional preparation period in the example demonstrated here, the STEP function affords a general editing tool for spectral simplification and can be applied to a range of experiments.

Ionophores↗
PubMed2003

Unique features in the C-terminal domain provide caltractin with target specificity.

Caltractin (centrin) is a member of the calmodulin (CaM) superfamily of EF-hand calcium-binding proteins. It is an essential component of the centrosomal structures in a wide range of organisms. Caltractin and calmodulin apparently function in distinct calcium signaling pathways despite substantial sequence similarity. In an effort to understand the structural basis for such differences, the high-resolution three-dimensional solution structure of the complex between the Ca(2+)-activated C-terminal domain of Chlamydomonas reinhardtii caltractin (CRC-C) and a 19 residue peptide fragment comprising the putative cdc31p-binding region of Kar1p (K(19)) has been determined by multi-dimensional heteronuclear NMR spectroscopy. Formation of the complex is calcium-dependent and is stabilized by extensive interactions between CRC-C and three key hydrophobic anchors (Trp10, Leu13 and Leu14) in the peptide as well as favorable electrostatic interactions at the protein-peptide interface. In-depth comparisons have been made to the structure of the complex of Ca(2+)-activated calmodulin and R(20), the CaM-binding domain of smooth muscle myosin light-chain kinase. Although the overall structures of CRC and CaM domains in their respective complexes are very similar, differences in critical regions in the sequences of these proteins and their targets lead to clear differences in the complementarity of their respective binding surfaces. These subtle differences reveal the structural basis for the Ca(2+)-dependent regulation of distinct cellular signaling events by CRC and CaM.

Amino Acid Sequence↗
PubMed2003

Different effects of topical prazosin and pilocarpine on uveoscleral outflow in rabbit eyes.

PURPOSE: To investigate the effects of topical prazosin and pilocarpine on uveoscleral outflow (Fu) in rabbits. METHODS: Sixteen rabbits were randomly divided into the control group (5 rabbits, only topical application of normal salino in the right eye of each rabbit), Prazosin (PZ) treated group (6 rabbits, only 0.1% Prazosin eyedrop 0.1% in the right eye of each one) and Pilocarpine (PC) treated group (5 rabbits, 1% Pilocarpine eye drop in each right eye). Intraocular pressure (IOP) of bilateral eyes of each rabbit was measured before and 1 h after topical application of the eye drop. And the bilateral eyes were perfused with Fluorescein-isothiocyanate bovine serum albumin (FITC-BSA) as the tracer into the anterior chamber of each rabbit for 30 min at 90 min after topical treatment. Then the rabbits were killed for Fu measurement. RESULTS: IOP of PZ-treated eyes decreased [(0.71 +/- 0.07)kPa] in 1 hour after PZ application. IOP of PC-treated eyes decreased [(0.70 +/- 0.08)kPa] in 1 hour after PC application. The average value of Fu was (0.176 +/- 0.048) microliter/min in control eyes. The average value of Fu in PZ-treated eyes was (0.339 +/- 0.018) microliter/min. The average value of Fu in PC-treated eye was (0.123 +/- 0.022) microliter/min. CONCLUSION: Both of PZ and PC can decrease IOP in rabbits following topical application. Topical PZ can increase Fu in rabbits and this is one of the mechanisms of PZ-induced IOP decrease. Topical PC can prevent Fu. PC decreases IOP not through uveoscleral pathway. This study demonstrates that uveoscleral pathway plays an important role in aqueous humor drainage. PZ may be a novel drug for decreasing IOP. FITC-BSA is an effective tracer for studying uveoscleral pathway.

Administration, Topical↗
PubMed2002

Structural independence of the two EF-hand domains of caltractin.

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.

Amino Acid Sequence↗