Biomedical subjects

D S Yang

Publications and source records attributed to D S Yang.

At least 37 records · Page 2Linked to original sources

PubMed1999

Studies of a putative ice-binding motif in winter flounder skin-type anti-freeze polypeptide.

Winter flounder contains two distinct anti-freeze protein isoforms, which are the liver-type extracellular anti-freeze proteins and the skin-type intracellular anti-freeze protein. The skin-type anti-freeze proteins exhibit lower anti-freeze activities than the liver-type isoforms and this might be due to their lacking complete ice-binding motifs. One of the skin-type anti-freeze proteins, skin-type anti-freeze protein-3, does contain putative overlapping ice-binding motifs with the sequences '-K-DT-' and '-DT-K-'. Synthetic anti-freezes containing 0-3 repeats of the '-DT-K-' motif were tested for stability and activity. Loss of the single '-DT-K-' of skin-type anti-freeze protein-3 increases the anti-freeze activity and increasing the number of motifs to two or three lowers the activity. The decrease in activity with an increasing frequency of the motif correlates with a decrease in the helical content of these peptides at 0 degrees C.

Animals↗

PubMed1999

Improved process for production of recombinant yeast-derived monomeric human G-CSF.

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secreted efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents.

Biopolymers↗

PubMed1999

Apolipoprotein E promotes the binding and uptake of beta-amyloid into Chinese hamster ovary cells in an isoform-specific manner.

The epsilon4 allele of apolipoprotein E gene is a major risk factor for Alzheimer's disease. However, the mechanism by which the E4 isoform of apolipoprotein E increases the risk of Alzheimer's disease is poorly understood. To determine whether the isoform-specific effects of apolipoprotein E may be mediated via clearance of bound beta-amyloid, we examined the uptake of beta-amyloid 1-40 into Chinese hamster ovary cells in the presence or absence of the apolipoprotein E isoforms E2, E3 and E4. Apolipoprotein E2 and E3 treatments were associated with higher association of beta-amyloid with cells as compared to treatment with E4. Heparin blocked the association of beta-amyloid with cells, as did an antibody to one of the apolipoprotein E receptors (the low-density lipoprotein receptor-related protein). Thus, the apolipoproteins E2 and E3, but not E4, may play important roles in the clearance of beta-amyloid from the extracellular space via the low-density lipoprotein receptor-related protein.

Amyloid beta-Peptides↗

PubMed1999

Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides.

Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-'. In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs. The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs. However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs. These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.

Amino Acid Sequence↗

PubMed1999

New clothes for amyloid enhancing factor (AEF): silk as AEF.

Amyloid enhancing factor (AEF) is an activity that appears naturally during the course of persistent inflammation and precedes, by 24-48 h, AA amyloid deposition in appropriate murine models. AEF is defined by its biological properties, namely, when administered intravenously or intraperitoneally to a mouse, it primes the recipient for the rapid induction of AA amyloid when they are given an inflammatory stimulus. Available evidence indicates that AEF is protein in nature, but a specific molecular species (if a singular species exits) has not been identified. Past work (Ganowiak et al., Biochem. Biophys. Res. Commun. 199:306-312, 1994) has shown that AEF activity may be imparted to two different proteins (IAPP and beta-protein) provided each is organized in the form of an amyloid fibril. Since a characteristic property of proteins in amyloid fibrils is their beta-sheet organization, one possibility is that AEF activity, in part, depends on such organization, and other proteins with such properties may also have AEF activity. To investigate this possibility, silk, a protein which contains substantial beta-sheet content, was denatured in LiSCN and allowed to renature slowly under reducing conditions to form a gel. The denatured silk preparation was then sonicated thoroughly to permit intravenous injection and assessed for AEF activity. The modified silk, presented as small fibrils in a beta-sheet conformation as assessed by electron microscopy and circular dichroism, respectively. This silk at 0-50 micrograms/animal was administered intravenously as "AEF" followed immediately by subcutaneous AgNO3 as the inflammatory stimulus. Six days later the spleens were examined for the presence of AA amyloid and following Congo red staining, the amount of amyloid quantified by image analysis. Modified silk without an inflammatory stimulus, and non-sonicated modified silk, failed to induce AA amyloid. Sonicated modified silk followed by AgNO3 induced large quantities of splenic AA amyloid in a dose dependent fashion. Modified silk in quantities as small as 1-5 micrograms/animal can function as AEF. The AEF properties of the modified silk were stable at 4 degrees C for at least 4 weeks (the longest period tested). This procedure may provide a means of standardizing AEF preparations.

Amyloid↗

PubMed1999

[Effect of selective lesions in hypothalamus on bilateral synchronized bursts of rat oxytocin neurons].

In order to clarify the location of the center for synchronized milk-ejection bursts of magnocellular oxytocin neurons in the hypothalamus, the bursts of these neurons were recorded extracellularly in lactating rats with selectively-cutting lesions of the middle brain or hypothalamus. Results showed that unilateral transection of the middle midbrain above the ventral tegmentum did not block the synchronized bursts on both sides; however, the synchronized bursts disappeared after unilateral transection through the middle of the medial hypothalamus. These results suggest that the area from the middle part of the midbrain to that of the hypothalamus does play a crucial role in the synchronized milk-ejection burst.

Animals↗

PubMed1998

Skin-type antifreeze protein from the shorthorn sculpin, Myoxocephalus scorpius. Expression and characterization of a Mr 9, 700 recombinant protein.

A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, alpha-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is alpha-helical at 0 degreesC and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing.

Amino Acid Sequence↗

PubMed1998

The ice-binding site of Atlantic herring antifreeze protein corresponds to the carbohydrate-binding site of C-type lectins.

The type II antifreeze proteins (AFPs) of smelt and Atlantic herring are homologous to the carbohydrate-recognition domains (CRDs) of Ca2+-dependent (C-type) animal lectins and, like these lectins, acquire a stable and active structure upon binding Ca2+ ions. In the C-type lectin CRD, the carbohydrate-binding site is located at a Ca2+-binding site. Site-directed mutagenesis was used to test the hypothesis that the ice-binding site of the type II AFP corresponds to the carbohydrate-binding site of the lectins. To disrupt this site in the herring AFP without perturbing the Ca2+-dependent protein fold, a double mutant was constructed that changed the Ca2+- and carbohydrate-binding motif from the galactose-type of wild-type AFP containing the sequence Gln-Pro-Asp to a mannose-type that has the sequence Glu-Pro-Asn and is also known to bind Ca2+. The mutant AFP exhibited proper Ca2+ binding, folding, and stability as demonstrated by ruthenium red staining, proteolysis protection assays, and CD spectroscopy. However, it showed no antifreeze activity (thermal hysteresis) and did not alter ice crystal morphology to form bipyramidal crystals as does the active wild-type AFP. These results demonstrate that the ice-binding site of the herring type II AFP corresponds to the carbohydrate-binding site of the C-type lectin CRDs and further suggest that this ice-binding function evolved from the carbohydrate-binding site of a preexisting C-type lectin.

Amino Acid Sequence↗

PubMed1998

Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration.

The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1-10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1beta. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1beta fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF.

Biotechnology↗

PubMed1998

Identification of the ice-binding surface on a type III antifreeze protein with a "flatness function" algorithm.

Antifreeze proteins (AFPs) adsorb to surfaces of growing ice crystals, thereby arresting their growth. The prevailing hypothesis explains the nature of adsorption in terms of a match between the hydrophilic side chains on the AFP's ice-binding surface (IBS) and the water molecules on the ice surface. The number and spatial arrangement of hydrogen bonds thus formed have been proposed to account, respectively, for the binding affinity and specificity. The crystal structure of a type III AFP from ocean pout (isoform HPLC-3) has been determined to 2.0-A resolution. The structure reveals an internal dyad motif formed by two 19-residue, loop-shaped elements. Based on of the flatness observed on the type I alpha-helical AFP's IBS, an automated algorithm was developed to analyze the surface planarity of the globular type III AFP and was used to identify the IBS on this protein. The surface with the highest flatness score is formed by one loop of the dyad motif and is identical to the IBS deduced from earlier mutagenesis studies. Interestingly, 67% of this surface contains nonpolar solvent-accessible surface area. The success of our approach to identifying the IBS on an AFP, without considering the presence of polar side chains, indicates that flatness is the first approximation of an IBS. We further propose that the specificity of interactions between an IBS and a particular ice-crystallographic plane arises from surface complementarity.

Algorithms↗

PubMed1998

[Optokinetic nystagmus (OKN) eye-movement mediated by direct and indirect pathways].

Nasal or temporal side of the cat's retina was stimulated with nasalward and temporalward moving patterns for investigating the neural control of the optokinetic nystagmus in the direct and indirect pathways of OKN. The experimental data demonstrated that there was a nasalward directional preference in cat's OKN system under both close loop and open loop conditions, and the nasalward OKN gain evoked by stimulation of nasal retina was much higher than that at temporal retina. This indicates that OKN nasalward preference is mainly derived from the nasal retina, i.e. the direct pathway of OKN dominates the indirect pathway in cat's OKN system. The gains of temporalward and nasalward OKN from temporal retina are much less than those of nasalward OKN from nasal retina. It is suggested that the indirect pathway of OKN in the cat only possesses a supplementary function, which probably plays an important role in temporalward OKN eye-movements in relation to binocular vision.

Animals↗

PubMed1997

Gem-dialkyl succinic acids: a novel class of inhibitors for carboxypeptidases.

gem-Dimethylsuccinic acid and its higher homolog, 2-methyl-2-ethylsuccinic acid (MESA) are highly potent inhibitors of both carboxypeptidase A (CPA) and B. The inhibition constant of MESA for CPA (0.11 microM for the racemic mixture) is remarkable considering the relatively simple structure of the compound. The molecular feature which is crucial for high affinity binding to both carboxypeptidases appears to be the nonpolar gem-dialkyl locus. The structure of the complex between MESA and CPA has been determined by X-ray crystallography to 2.0 A resolution and shows the R enantiomer of the inhibitor to be bound in a generally substrate-like manner. The carboxymethyl group is coordinated to the Zn ion in the active site, and the gem-dialkyl locus corresponds in position to the alpha-carbon of the C-terminal amino acid in a peptide substrate. The methyl group of the inhibitor occupies a cavity in the enzyme which is apparently not filled upon substrate-binding. We postulate that this cavity (the alpha-methyl hole) is designed to allow the proximal Glu-270 residue to undergo a critical movement during catalysis. The hydrophobic nature of the above cavity may play a role in modulating the reactivity of this residue. These results suggest that similar cenophilic(empty-loving) inhibitors may be found for other enzymes.

Animals↗

PubMed1997

Development of the enkephalin-, neurotensin- and somatostatin-like (ENSLI) amacrine cells in the chicken retina.

The development of the enkephalin-, neurotensin- and somatostatin-like immunoreactive (ENSLI) amacrine cells in the chicken retina has been investigated by radioimmunoassay (RIA) and immunocytochemistry (ICC). By RIA, enkephalin-like immunoreactivity (ENK-LI) was detected at embryonic day (E) 5 at only very low levels, which gradually increased until E17. From E18 to E21, there was a relatively rapid increase in ENK-LI levels, and just after hatching, there was a very steep rise. By ICC, the cell bodies of the ENSLI amacrine cells were first detected in the inner nuclear layer on E18, with no immunostaining in the inner plexiform layer (IPL). On E21, more cells were detected and processes in the IPL were visible, but detailed arborisations were not clear. On postnatal day (P) 1, the ENSLI amacrine cells showed a morphology similar to that in mature retina in both the density of cell bodies and the ramification pattern of processes. Antibodies to neurotensin and somatostatin revealed a similar developmental pattern. Thus, the three peptides appear to follow a similar developmental pattern in the ENSLI amacrine cells, suggesting that the three peptides respond similarly to developmental stimuli, just as they are released in parallel in response to physiological stimulation from mature ENSLI amacrine cells. After hatching, higher levels of ENK-LI were detected by RIA and more ENSLI amacrine cell bodies and processes were detected by ICC in animals kept in the light than in those kept in the dark. In retinas kept in the light for 12 h, it was found that immunoreactive processes in the IPL formed strongly stained patches, but this was not observed in retinas kept in the dark for 12 h.

Animals↗

PubMed1997

Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases.

Bacterial resistance to aminoglycoside antibiotics is almost exclusively accomplished through either phosphorylation, adenylylation, or acetylation of the antibacterial agent. The aminoglycoside kinase, APH(3')-IIIa, catalyzes the phosphorylation of a broad spectrum of aminoglycoside antibiotics. The crystal structure of this enzyme complexed with ADP was determined at 2.2 A. resolution. The three-dimensional fold of APH(3')-IIIa reveals a striking similarity to eukaryotic protein kinases despite a virtually complete lack of sequence homology. Nearly half of the APH(3')-IIIa sequence adopts a conformation identical to that seen in these kinases. Substantial differences are found in the location and conformation of residues presumably responsible for second-substrate specificity. These results indicate that APH(3') enzymes and eukaryotic-type protein kinases share a common ancestor.

Aminoglycosides↗

PubMed1997

Characterization of the binding of amyloid-beta peptide to cell culture-derived native apolipoprotein E2, E3, and E4 isoforms and to isoforms from human plasma.

The epsilon 4 allele of apolipoprotein E (apoE, protein; APOE, gene) is a major risk factor for Alzheimer's disease (AD). Genetically, the frequency of the epsilon 4 allele is enriched in early-onset sporadic, late-onset familial, and common late-onset sporadic AD. ApoE is found in the extracellular amyloid-beta (A beta) deposits that are characteristic features of AD. In this study, we examined the interaction between A beta and apoE isoforms. The apoE isoforms used in this study were either produced by stably transfected Chinese hamster ovary cells (CHO) or were from human plasma. We report that when similar concentrations of the apoE isoforms were used, native nonpurified apoE3 from recombinant CHO-derived sources bound A beta, but apoE4 did not. In fact, in our system, binding of recombinant apoE4 to A beta was never detectable, even after incubation for 4 days. Furthermore, using the same assay conditions, native apoE2, like apoE3, binds A beta avidly. Furthermore, when human plasma apoE isoforms are tested in A beta binding experiments, apoE3 bound A beta more avidly than apoE4, and a major apoE/A beta complex (the 40-kDa form) was observed with plasma apoE3 but not apoE4. These data extend our understanding of apoE isoform-dependent binding of A beta by associating apoE2 with efficient apoE/A beta complex formation and demonstrate that native apoE3 (whether recombinant or derived from human plasma) forms sodium dodecyl sulfate-stable apoE/A beta complexes more readily than native apoE4. The different A beta-binding properties of native apoE4 versus native apoE3 provide insight into the molecular mechanisms by which the APOE epsilon 4 allele exerts its risk factor effects in AD.

Amyloid beta-Peptides↗

PubMed1997

A light-driven rhythm in neurotensin-like immunoreactivity in the chicken retina.

PURPOSE: To determine if the pattern of release of neurotensin from the enkephalin-, neurotensin- and somatostatin-like immunoreactive amacrine cells in response to light and dark is the same as that of the enkephalins and somatostatin. METHODS/RESULTS: Both the enkephalins and somatostatin are released at high rates in the dark and at lower rates in the light, and these rate changes are reflected in increasing intracellular levels of the peptides in vivo in the light and decreasing levels in the dark. The levels of neurotensin-like immunoreactivity show a similar diurnal light-driven and non-circadian rhythm in vivo. CONCLUSION: This implies that the actual release rates of neurotensin follow the same patterns as those demonstrated in vitro for the enkephalins and somatostatin.

Adaptation, Ocular↗

PubMed1996

Ca2+-dependent antifreeze proteins. Modulation of conformation and activity by divalent metal ions.

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. The type II fish AFPs of Atlantic herring and smelt are unique among known AFPs in their requirement of a cofactor for antifreeze activity. These AFPs are homologous with the carbohydrate-recognition domains of Ca2+-dependent (C-type) lectins and require Ca2+ for their activity. To investigate the role of metal ions in the structure and function of type II AFPs, the binding of Ca2+ and other divalent cations to herring AFP was investigated. Binding studies using 45Ca2+ demonstrated that the AFP has a single Ca2+-binding site with a Kd of 9 microM. Proteolysis protection studies and measurement of antifreeze activity revealed a conformational change from a protease-sensitive and inactive apoAFP to a protease-resistant active AFP upon Ca2+ binding. Other divalent metal ions including Mn2+, Ba2+, and Zn2+ bind at the Ca2+-binding site and induce a similar change. A saturatable increase in tryptophan emission intensity at 340 nm also occurred upon Ca2+ addition. Whereas antifreeze activity appeared normal when Ca2+ or Mn2+ were bound, it was much lower in the presence of other metal ions. When Ba2+ was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca2+ and, consequently, adopts a conformation that is essential for its ice-binding activity.

Amino Acid Sequence↗

PubMed1996

Structure determination of a lone alpha-helical antifreeze protein from winter flounder.

The X-ray crystal structure of a lone alpha-helical antifreeze protein from winter flounder has been determined to 1.5 A using a combination of molecular-replacement and isomorphous-replacement techniques. Molecular replacement involved a multiparameter search using X-PLOR with two 37-mers of alanine in idealized alpha-helical conformations as the search models. Identified were a large number of potential solutions from which the correct solution was not distinguishable. Commitment of the top 1620 solutions to cycles of rigid-body, positional and simulated-annealing refinement identified the correct solution by a small margin in R factor. Low-resolution electron-density maps generated with phasing information from TbNO(3) and LaNO(3) derivatives were consistent with the top molecular-replacement solution. These derivatives also provided a means to filter and compare the large number of other molecular-replacement solutions with reasonable R factor statistics. The structure-solution strategy described herein may prove useful for the determination of other relatively simple alpha-helical X-ray structures.

Journal Article↗