High-frequency representation of a single VH gene in the expressed human B cell repertoire.
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B-Lymphocytes↗
Biomedical subjects
C Huang
Publications and source records attributed to C Huang.
Comparison of phospholipase D activity in vasopressin- and phorbol ester-stimulated fibroblasts.
Phospholipase D (PLD) activation by vasopressin (VP) was compared to activation by TPA in REF52 cells prelabeled with [3H]glycerol and [14C]myristic acid. Upon VP-treatment, the formation of [3H] and [14C]phosphatidic acid (PA) and phosphatidylethanol (PEt) was accompanied by the loss of radioactivity from PC and PI. However, upon TPA-treatment, radioactivity was lost from PC only. No significant changes of phosphatidylethanolamine and phosphatidylserine were detected in the same samples. The inclusion of 5 microM staurosporine for 10 min diminished the production of [3H]PEt and [14C]PEt by 27% and 53% in VP-treated cells, and by 100% and 75% in TPA-treated cells, respectively. Adding 1 mM EGTA to chelate extracellular Ca2+ inhibited [3H]PEt by approximately 31% and [14C]PEt by 17% after VP-stimulation. In contrast, EGTA had no effect on TPA-stimulation. The data suggest that REF52 cells contain dual PLD activities. The first is stimulated only by VP, requires Ca2+ and hydrolyzes PI. The second is stimulated by both TPA and VP, activated by protein kinase C and hydrolyzes PC.
Alkaloids↗
Calorimetric studies of fully hydrated phosphatidylcholines with highly asymmetric acyl chains.
We have semi-synthesized 52 molecular species of saturated diacyl mixed-chain phosphatidylcholines. All 52 phosphatidylcholine molecules are highly asymmetrical with delta C/CL values in the range of 0.43-0.63. The aqueous dispersions of these phosphatidylcholines have been studied by the high-resolution differential scanning calorimetric (DSC) technique. Upon heating, the lipid dispersions prepared individually from these 52 phosphatidylcholines all exhibit a sharp, single, endothermic peak at a characteristic temperature or Tm, implying that the self-assembled lipid molecules in excess water undergo the mixed interdigitated gel to the liquid-crystalline phase transition. The Tm values obtained from aqueous lipid dispersions prepared from these mixed-chain phospholipids have been analyzed based on the molecular packing model of the mixed interdigitated bilayer, and a linear relationship between the Tm and (delta C)-1 for various phospholipids at a constant value of delta is observed. Based on these linear relationships, empirical equations are derived to predict the Tm values for highly asymmetrical mixed-chain phosphatidylcholines with delta C/CL values in the range of 0.43-0.63. The predictive power of these empirical equations is shown to be very good, since a comparison between the predicted and the experimental data indicates that the largest relative error in Kelvin is only 0.4%. A table containing 81 predicted Tm values for highly asymmetrical mixed-chain phosphatidylcholines is presented. The definitions of the various structural parameters such as delta C, CL, delta C/CL and delta are given in the text.
Calorimetry, Differential Scanning↗
High-frequency representation of a single VH gene in the expressed human B cell repertoire.
Idiotype (Id) 16/6 marks a variable (V) region structure that occurs frequently in the human immunoglobulin repertoire. The basis of the Id has been traced to a germline heavy chain gene segment, VH18/2 (VH26). To pursue the molecular basis for the frequency of Id 16/6, we have analyzed polymerase chain reaction-generated C mu, C gamma, and VH3 family V gene libraries derived from the circulating and tonsillar B cells of four normal individuals and from the B cells of two patients with active systemic lupus erythematosus (SLE). The frequency of VH18/2 in these libraries was compared with three control VH genes, VH56P1, VH21/28, and VHA57. Plaque lifts from C mu and C gamma VH cDNA libraries were screened with gene-specific oligonucleotide probes. The frequency of VH18/2 ranged from 4 to 10% of JH+ plaques (two of five times that of control VH genes). In four VH3 family-specific libraries derived from rearranged DNA, VH18/2 represented 19-33% of VH3+ plaques. Hybridizing VH18/2 plaques were 98-100% homologous to the germline VH gene; mutations when present were often in framework 3. Extensive variation was seen in the complementarity determining region 3 sequences of these rearranged V genes. The high frequency of VH18/2 expression in the B cell repertoire was confirmed by sequencing randomly picked JH+ plaques. In two patients with active SLE the frequency of use of VH18/2 was not greater than that observed in normal subjects. These results show that VH18/2 is overrepresented in the B cell repertoire of normal subjects and suggest that the immune repertoire may be dominated by relatively few V genes.
Amino Acid Sequence↗
Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii.
Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a 'yellow-in-the-dark' phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.
Algal Proteins↗
Dependence of the bilayer phase transition temperatures on the structural parameters of phosphatidylcholines.
Most saturated diacyl phosphatidylcholines C(X):C(Y)PC (saturated 1,2-diacyl-sn-glycero-3-phosphocholine with X carbons in the sn-1 acyl chain and Y carbons in the sn-2 acyl chain), in excess water, can self-assemble into lamellae which, upon heating, may undergo multiple thermotropic phase transitions at well-defined, discrete temperatures. The transition temperature corresponding to the main or the gel to liquid-crystalline phase transition (Tm) is known for many bilayers of fully hydrated phosphatidylcholines. In this study, we have analyzed the Tm values of 44 molecular species of phosphatidylcholines in terms of their structural and packing characteristics in the gel-state bilayer. Two general equations are thus derived: Tm = 162.26-3651.71 (1/N) - 88.42 (delta C/N) for C(X):C(Y)PC with X > or = Y, and Tm = 157.68-3525.44 (1/N) - 93.28 (delta C/N) for C(X):C(Y)PC with X < Y. Here, N is the minimal hydrophobic thickness of the dimeric C(X):C(Y)PC in the gel-state bilayer and delta C is the effective chain length difference between the sn-1 and sn-2 acyl chains for the monomeric C(X):C(Y)PC in the gel-state bilayer. The advantage of these two equations in predicting the Tm values for phosphatidylcholines with delta C/CL values in the range of 0.07 to 0.40 is their simplicity. A figure containing a total of 173 calculated Tm values is also presented.
Computer Simulation↗
Energy-minimized structures and packing states of a homologous series of mixed-chain phosphatidylcholines: a molecular mechanics study on the diglyceride moieties.
Phosphatidylcholines or C(X):C(Y)PC, quantitatively the most abundant lipids in animal cell membranes, are structurally composed of two parts: a headgroup and a diglyceride. The diglyceride moiety consists of the glycerol backbone and two acyl chains. It is the wide diversity of the acyl chains, or the large variations in X and Y in C(X):C(Y)PC, that makes the family of phosphatidylcholines an extremely complex mixture of different molecular species. Since most of the physical properties of phospholipids with the same headgroup depend strongly on the structures of the lipid acyl chains, the energy-minimized structure and steric energy of each diglyceride moiety of a series of 14 molecular species of phosphatidylcholines with molecular weights identical to that of dimyristoylphosphatidylcholine without the headgroup are determined in this communication by molecular mechanics (MM) calculations. Results of two types of trans-bilayer dimer for each of the 14 molecular species of phosphatidylcholines are also presented; specifically, the dimeric structures are constructed initially based on the partially interdigitated and mixed interdigitated packing motifs followed subsequently by the energy-minimized refinement with MM calculations. Finally, tetramers with various structures to model the lateral lipid-lipid interactions in a lipid bilayer are considered. Results of laborious MM calculations show that saturated diacyl C(X):C(Y)PC with delta C/CL values greater than 0.41 prefer topologically to assemble into tetramers of the mixed interdigitated motif, and those with delta C/CL values less than 0.41 prefer to assemble into tetramers with a repertoire of the partially interdigitated motif. Here, delta C/CL, a lipid asymmetry parameter, is defined as the normalized acyl chain length difference between the sn-1 and sn-2 acyl chains for a C(X):C(Y)PC molecule; an increase in delta C/CL value is an indication of increasing asymmetry between the two lipid acyl chains. These computational results are in complete accord with the calorimetric data presented previously from this laboratory (H-n. Lin, Z-q. Wang, and C. Huang. 1991. Biochim. Biophys. Acta. 1067:17-28).
Animals↗
Role of haematocrit in mediating the actions of chronic erythropoietin treatment on blood pressure and renal haemodynamics in the rat.
1. This investigation aimed to study the effect of chronic administration of human recombinant erythropoietin on haematocrit, blood pressure, renal cortical and papillary resistances and vascular responsiveness to vasoconstrictor agents. 2. Rats were treated with placebo or 25, 50 or 100 units/kg erythropoietin subcutaneously, every other day for 3 weeks. Animals were then anaesthetized with sodium pentobarbitone and were prepared for laser-Doppler flowmetry measurement in the renal cortex and papilla. 3. Haematocrit in the placebo-treated group was 48.0 +/- 0.5% and was raised to 52.5 +/- 0.7, 55.9 +/- 0.8 and 62.4 +/- 1.1% (all P < 0.05) by the chronic administration of 25, 50 and 100 units/kg doses of the hormone, respectively. Blood pressure was 107 +/- 1 mmHg in the placebo-treated group and was elevated to 116 +/- 2 and 130 +/- 1 mmHg (both P < 0.05), respectively, by the two highest doses of erythropoietin. Cortical and papillary perfusions were reduced at the highest dose of erythropoietin, but calculated resistances were increased by 15 and 40% (P < 0.05) at 50 and 100 units/kg doses of the hormone, respectively. 4. Infusion of the vasopressor hormones vasopressin and phenylephrine caused increases in blood pressure and decreases in renal cortical and papillary perfusion, the magnitudes of which were only marginally changed by the highest dose of the erythropoietin. Angiotensin II increased blood pressure and decreased cortical perfusion, and the magnitudes of these responses were unchanged by the chronic treatment with erythropoietin. 5. Acute graded increases in haematocrit resulted in significantly (P < 0.05) raised blood pressure above a value of 58%.(ABSTRACT TRUNCATED AT 250 WORDS)
Angiotensin II↗
Differential regulation of the CYP11A1 (P450scc) and ferredoxin genes in adrenal and placental cells.
The regulation of the genes encoding cholesterol side-chain cleavage enzyme (P450scc) and ferredoxin, two components in the first step of steroid synthetic pathways, was studied by RNA analyses of endogenous and transfected genes. cAMP rather than calcium was the major secondary messenger that stimulated expression of both P450scc and ferredoxin genes in human placental JEG-3 cells. The effect of cAMP on P450scc expression was abolished by cycloheximide in JEG-3 cells, but it was superinduced in mouse adrenal Y1 cells. For ferredoxin expression, both reagents have synergistic effect in Y1 and JEG-3 cells. To test the mechanism of regulation, DNA segments containing regulatory elements of the P450scc and ferredoxin genes were connected to reporter genes and analyzed in cotransfection experiments. The results showed that the proximal cAMP-responsive sequences of both P450scc and ferredoxin genes were stimulated by cAMP early in both Y1 and JEG-3 cells, requiring no new protein synthesis. This indicates a common mechanism for the regulated expression of both genes. P450scc possessed an additional upstream cAMP-responsive sequence that also responded to cAMP induction in a different manner from the proximal element. The presence of additional upstream regulatory elements makes it possible for the P450scc gene to be further regulated.
Adrenal Glands↗
Feynman path integral for an infinite potential barrier.
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Journal Article↗
Stimulation of prolactin secretion in the pig: central effects of relaxin and the antiprogesterone RU 486.
Our previous study has shown that oral administration of a potent progesterone antagonist, RU 486, caused a marked elevation of plasma concentrations of both PRL and progesterone in hysterectomized pigs bearing aging corpora lutea. Hysterectomized pigs (hysterectomy performed on day 8; estrus = day 0) were subjected to cranial surgery for chronic placement of a head-mounted stereotaxic apparatus for intracerebroventricular (icv) administration of relaxin (300 U once daily on days 111 and 113; n = 6) and RU 486 (4 mg once daily on days 111, 113, and 115; n = 5) to test whether relaxin and RU 486 exert their actions within the central nervous system and/or pituitary gland to affect PRL and GH secretion. Control pigs (n = 3) received icv injection of vehicle. Intensive blood sampling revealed that icv injection of relaxin on day 111 markedly increased the plasma PRL concentration from 8 to 38 ng/ml within 10 min (P < 0.01). An identical icv injection of relaxin on day 113 caused only a modest increase in PRL, but the overall mean concentration of PRL after relaxin treatment was greater than that before treatment (14 vs. 8 ng/ml; P < 0.05). Intracerebroventricular injection of RU 486 on day 111 greatly elevated plasma PRL. The increase in PRL lasted more than 2 h, with several peak increases of 18-29 ng/ml (P < 0.01). The PRL response to subsequent icv infusion of RU 486 on days 113 and 115 was blunted, but the overall mean concentration of PRL (14 ng/ml) after icv injection of RU 486 remained greater (P < 0.01) than that before treatment (9 ng/ml). In contrast, PRL concentrations in the control group remained unchanged after injection. Plasma concentrations of GH, relaxin, and progesterone were significantly altered in neither hormone- nor vehicle-treated groups during this brief period of sequential blood sampling. This study provides strong evidence that relaxin has a central role in modulating PRL secretion in the pig. In addition, the antagonistic effects on progesterone receptor by RU 486 in the central nervous system and/or pituitary gland caused an abrupt increase in PRL secretion in these hysterectomized gilts.
Animals↗
Stimulation of collagen secretion by relaxin and effect of oestrogen on relaxin binding in uterine cervical cells of pigs.
The role of relaxin and oestrogen in the remodelling of connective tissue was investigated by testing collagenase-dispersed cells (3 x 10(5) cells per well) from the uterine cervix of gilts for relaxin binding and collagen secretion. Relaxin-binding sites on these cells were quantified by specific binding of a saturating dose of 125I-labelled monotyrosyl relaxin at optimal conditions. Oestrogen at doses from 0.4 to 50 ng ml-1 increased relaxin binding in a time- and dose-dependent manner. Scatchard plot analysis exhibited curvilinearity, which suggested two classes of relaxin-binding sites. The addition of relaxin (0, 100, 500 ng ml-1) alone (P < 0.05) or in combination with oestrogen (oestradiol benzoate: 0, 50, 250 ng ml-1) increased protein secretion into the culture medium. Hydroxyproline concentration (as an index of collagen) in the medium was increased (P < 0.05) only in the presence of both relaxin and oestrogen. Actinomycin D (500 ng ml-1) and cycloheximide (500 ng ml-1) inhibited hydroxyproline secretion induced by combined relaxin and oestrogen treatment. Dibutyryl cyclic adenosine 3',5'-monophosphate (dibutyryl cAMP: 0, 0.1, 1.0, 5.0 mmol l-1) was a potent stimulator of hydroxyproline secretion. These results indicate that relaxin, probably via a cAMP pathway, stimulates hydroxyproline secretion in the presence of oestrogen through a protein- and RNA-synthesis dependent process. Oestrogen plays a role in augmenting the sensitivity of uterine cervical cells to relaxin in the pig.
Animals↗
Effects of aprotinin in platelet aggregation and cytosolic free calcium in swine platelets.
Aprotinin inhibited platelet aggregation induced by thrombin (0.25 U.ml-1) with IC50 200 kIU.ml-1, and inhibited the rise of cytosolic free calcium concentration in platelets stimulated by thrombin (0.1 U.ml-1) in the absence and in the presence of Ca2+ 0.5 mmol.L-1 (IC50 117 and 50 kIU.ml-1, respectively), but had no effect on the amounts of actin and myosin heavy chain associated with cytoskeletons. These suggest that aprotinin is an anti-platelet agent and may exert its action through inhibiting the Ca2+ flux.
Animals↗
High level expression of human prourokinase cDNA in Chinese hamster ovary cells.
We have used Chinese hamster ovary (CHO) cells to express high levels of human prourokinase gene cDNA with recourse to construction of good expression vector, the improvement of transfection technique and gene coamplification. First, we constructed expression plasmid pMG10102 by placing pro-UK cDNA under the control of SR alpha promoter/SV40 polyadenylation signals and expressed it transiently in COS-7 cells. Expression level was about 5 times higher than with SV40 early promoter. Linear plasmids pMG10102 and pSV2-dhfr were then cotransfected into CHO-dhfr cells by calcium phosphorate coprecipitation and cells were cultured in selective medium. Twenty transformants expressing pro-UK were picked, the range of expression levels was 12.5-100IU/10(6) cells/day. When subjected to stepwise selection of methotrexate (MTX), the stable cell lines were obtained that secreted up to 400-500IU/10(6) cells/day. Western blot analysis showed that molecular weight of secreted recombinant pro-UK was the same as that of natural pro-UK, which is 52 kDa, and more than 60% of expression production was single chain urokinase (rscUK) without protease inhibitor in medium.
Animals↗
A computerized expert system for outcome-validated medical practice guidelines.
Our ongoing efforts in health services research have resulted in outcome-validated medical practice guidelines for common medical conditions. These practice guidelines have been shown to substantially reduce health care costs while maintaining quality of care. We have developed a computerized expert system from our practice guidelines which enhances the ease in which they can be implemented by Utilization Management (UM) Coordinators, physicians, nurses and other health care providers.
Cost Control↗
[Quantitative study of transmitral flow by spectral Doppler echocardiography].
The aim of this study is to find an accurate and simple method for measuring the transmitral flow by pulsed Doppler echocardiography (PDE). Four parameters were measured: (1) anteroposterior dimension of the mitral valve annulus (MVA) (a); (2) mediolateral dimension of MVA (b); (3) a and b of MVA to calculate the area of MVA (elliptical method); (4) the area of the mitral valve orifice (MVO). The transmitral flow calculated by the use of (3) and (4) were compared with the aortic blood flow. The findings suggested that the MVA calculated with elliptical method is accurate and simple in measuring the transmitral flow.
Adolescent↗
High level expression of Shiga toxin B subunit of Shigella dysenteriae serotype 1 in Escherichia coli.
In this study the gene encoding the Shiga toxin (Stx) was cloned from the chromosomal DNA of Shigella dysenteriae serotype 1 (W30864). The Stx gene was located in a 4.5kb EcoRI fragment. The biological assay revealed that the hybrid plasmid pMGC001 containing the Stx gene could produce Shiga toxin. The amount of Stx was 16 times more than that produced by its parent. The cloned strain showed cytotoxic, entorotoxic and neurotoxic activities. The gene for Stx-B subunit was subcloned into a plasmid vector pJLA503 from the plasmid pMGC001. The B subunit could be expressed at high level in E. coli and has been purified in large quantities. The polyclonal and monoclonal antibodies against B subunit were raised. Western blot showed that they could react specifically to the B subunit.
Bacterial Toxins↗
Sequence analysis of the colonization factor antigen 1 gene of ETEC and observation of CFA/1 recombinant clone with electron microscopy.
The structure gene of the colonization factor antigen 1 (CFA/1) has been sequenced. The amino acid sequence of CFA/1 precursor deduced from the nucleotide sequence is composed of 170 amino acids. The first 23 amino acids are considered to be the signal peptide of the CFA/1 protein. On the basis of the nucleotide sequence, three amino acids are different from the protein sequence. Amino acid residues at positions 37, 76 and 97 are found to be Ala, Asp and Ser, instead of Val, Asn and Ala as determined by amino acid sequencing. The CFA/1 is very hydrophobic amino acids. Among the total 170 amino acids, 47% of them are hydrophobic amino acids. CFA/1 gene has a typical Shine-Dalgarno sequence and-10 sequence, but no 35 promoter sequence could be found. The G + C ratio of CFA/1 gene is 40%. Observation of negatively stained preparations of the culture with electron microscopy showed that no fimbriae were found on E. coli C600 strain itself, but the cell carrying the plasmid showed thick pili on its surface.
Amino Acid Sequence↗