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Biomedical subjects

P S Ho

Publications and source records attributed to P S Ho.

At least 19 recordsLinked to original sources
PubMed1999

The intrinsic structure and stability of out-of-alternation base pairs in Z-DNA.

Alternating pyrimidine-purine sequences typically form Z-DNA, with the pyrimidines in the anti and purines in the syn conformations. The observation that dC and dT nucleotides can also adopt the syn conformation (i.e. the nucleotides are out-of-alternation) extends the range of sequences that can convert to this left-handed form of DNA. Here, we study the effects of placing two adjacent d(G*C) base pairs as opposed to a single d(G*C) base pair or two d(A*T) base pairs out-of-alternation by comparing the structure of d(m5CGGCm5CG)2with the previously published structures of d(m5CGGGm5CG)*d(m5CGCCm5CG) and d(m5CGATm5CG)2. A high buckle and loss of stacking interactions are observed as intrinsic properties of the out-of-alternation base pairs regardless of sequence and the context of the dinucleotide. From solution titrations, we find that the destabilizing effect of out-of-alternation d(G*C) base pairs are identical whether these base pairs are adjacent or isolated. We can therefore conclude that it is these intrinsic distortions in the structure of the base pairs and not neighboring effects that account for the inability of out-of-alternation base pairs to adopt the left-handed Z conformation.

Base Pairing↗
PubMed1998

AcMNPV late expression factor-5 interacts with itself and contains a zinc ribbon domain that is required for maximal late transcription activity and is homologous to elongation factor TFIIS.

The late expression factor-5 gene (lef-5) of Autographa californica multinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione-S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a beta-glucuronidase (GUS) reporter gene under control of the late vp39 capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn2+ caused a reduction in activity similar to that when the domain was eliminated completely.

Alanine↗
PubMed1997

Growth hormone regulates ternary complex factors and serum response factor associated with the c-fos serum response element.

For insight into the mechanisms of gene regulation by growth hormone (GH), the regulation of transcription factors associated with the serum response element (SRE) located upstream of c-fos was examined. The SRE can mediate induction of reporter expression in response to GH. For insight into the mechanism by which GH regulates transcription factors, regulation of SRE-associated proteins by GH was examined. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2-10 min transiently increased binding of two complexes; binding returned to control values within 30 min. The two GH-stimulated complexes were supershifted by antibodies against the serum response factor (SRF), indicating that they contained SRF or an antigenically related protein. One of the GH-stimulated complexes was supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH was lost when the SRF binding site in the SRE was mutated, and mutation of either the SRF or TCF binding site altered the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibited the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression was mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulated the serine phosphorylation of Elk-1. The increase was evident within 10 min and subsided after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1 contributing to GH-promoted transcriptional activation via the SRE.

3T3 Cells↗
PubMed1997

The structures and relative stabilities of d(G x G) reverse Hoogsteen, d(G x T) reverse wobble, and d(G x C) reverse Watson-Crick base-pairs in DNA crystals.

We have solved the structures of the homoduplex d(Gm5CGCGCG)2, and the heteroduplexes d(GCGCGCG)/d(TCGCGCG) and d(GCGCGCG)/d(CCGCGCG). The structures form six base-pairs of identical Z-DNA duplexes with single nucleotides overhanging at the 5'-ends. The overhanging nucleotide from one strand remains stacked and sandwiched between the blunt-ends of two adjacent Z-DNA duplexes, while the overhanging base of the opposing strand is extra-helical. The stacked and the extra-helical bases from adjacent duplexes pair to form a distorted d(G x G) reverse Hoogsteen base-pair in the d(Gm5CGCGCG)2 homoduplex, and d(G x T) reverse wobble and d(G x C) reverse Watson-Crick base-pairs in the d(GCGCGCG)/d(TCGCGCG) and d(GCGCGCG)/d(CCGCGCG) heteroduplexes, respectively. Interestingly, only the d(G,T) and d(G x C) base-pairs were observed in the heteroduplexes, suggesting that both the d(G x T) reverse wobble and d(G x C) reverse Watson-Crick base-pairs are more stable in this crystal environment than the d(G x G) reverse Hoogsteen base-pair. To estimate the relative stability of the three types of reverse base-pairs, crystals were grown using various mixtures of sequences and their strand compositions analyzed by mass spectrometry. The d(G x C) reverse Watson-Crick base-pair was estimated to be more stable by approximately 1.5 kcal/mol and the d(G x T) reverse wobble base-pair more stable by approximately 0.5 kcal/mol than the d(G x G) reverse Hoogsteen base-pair. The step during crystallization responsible for discriminating between the strands in the crystal is highly cooperative, suggesting that it occurs during the initial nucleating event of crystal growth.

Computer Simulation↗
PubMed1997

Aetiology, morphology and body composition of infants born small for gestational age.

Infants born small for gestational age (SGA) are a heterogeneous group. Both the timing and duration of the intrauterine insult determine the physical condition and body composition of the infant at birth. Infants with symmetrical intrauterine growth retardation (IUGR) have a similar body composition at birth to weight-matched infants born appropriate for gestational age. However, these infants are more likely to remain shorter and lighter than normal infants. In contrast, infants with asymmetrical IUGR have reduced fat deposition but are more likely to exhibit catch-up growth during the first few months of life. The low mortality and morbidity rates in infants born SGA observed in recent studies are linked to their appropriate perinatal management, including adequate early nutritional support.

Body Composition↗
PubMed1997

Use of dual-energy X-ray absorptiometry for the measurements of small quantities of mineral.

The aim of the study was to assess the precision and the accuracy of whole-body dual-energy X-ray absorptiometry (DXA) measurements of low quantities of mineral. Six stillborn infants were measured three times in order to assess the precision in vivo. Powdered bone tablets were also measured three times separately or in compact groups (0.32-41.7 g of hydroxyapatite) by whole-body DXA. Mean correlation coefficients were 6.85 +/- 5.99 and 6.74 +/- 6.58% in vivo and in vitro, respectively. DXA-based estimates of the tablets were highly correlated with the actual bone mineral content (BMC; r = 0.99, p = 0.0001). The mean difference between DXA values and the reference values was 2.55 +/- 10.93%, but the agreement between DXA results and reference values rose to 3.99 +/- 3.49% for BMC above 10 g. Very small amounts of mineral can be measured by whole-body DXA, but assessment of an infant with a BMC less than 10 g should be made with care.

Absorptiometry, Photon↗
PubMed1996

Sequence-dependent effects of spermine on the thermodynamics of the B-DNA to Z-DNA transition.

Spermine has been shown to bind to and stabilize a number of altered DNA conformations, including left-handed Z-DNA. Here, we have quantitatively studied the effects of spermine on the negative supercoil-induced transition from B- to Z-DNA. We have determined the intrinsic association constants for and the effective number of ligands that bind to both B- and Z-DNA. The intrinsic affinity of spermine for Z-DNA is approximately 10 times higher for d(CA/TG) (KZP = 1.2 x 10(8) M-1) than for d(CG) dinucleotides (KZP = 1.5 x 10(7) M-1), and both are greater than that for B-DNA (KBP = 1.4 x 10(5) M-1). This accounts for the stabilization of Z-DNA by spermine. The number of spermine accommodated by Z-DNA (nZ) is sequence-dependent [nZ = 0.6 spermine per 18 d(CA/TG) dinucleotides and 2.3 for 12 d(CG) dinucleotides]. The value of nZ of < 1 was interpreted as evidence for negative cooperativity in spermine binding to d(CA/TG) dinucleotides. Thus, although d(CA/TG) sequences saturate at lower spermine concentrations, the ligand has an overall greater effect on the stability of d(CG) dinucleotides as Z-DNA. B-DNA accommodates more spermines per base pair than either sequence as Z-DNA. At higher concentrations (> 10 microM), spermine destabilizes Z-DNA. Using these parameters in a model for competitive spermine binding to B-DNA and Z-DNA, we can make predictions for how potential Z-DNA sequences found in the human genome are affected by cellular levels of superhelical density and spermine.

DNA↗
PubMed1996

Structure-function analysis of the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region palindromes.

Homologous regions (hrs), which are present at eight dispersed locations on the Autographa californica multinucleocapsid nuclear polyhedrosis virus genome, are composed of repeated imperfect palindromes within directly repeated sequences. Hrs act as transcriptional enhancers of RNA polymerase II-mediated transcription and as origins of DNA replication when incorporated into plasmids and tested in transient replication assays. To characterize the physical structure of these elements and to determine the role that mismatched nucleotides may play in hr function, oligonucleotides containing a consensus mismatched "imperfect" palindrome and a closely related "perfect" palindrome were synthesized. These sequences were cloned into individual plasmids and tested for their ability to form cruciform structures using nuclease P1 assays and two-dimensional (2-D) gel analyses of topoisomers. The perfect palindrome formed a cruciform structure and the energy requirement for its formation was predicted to occur under physiological conditions. In contrast, the construct containing an imperfect palindrome did not form a cruciform under these conditions. Both hr constructs were found to bind IE-1 in electrophoresis mobility shift assays and act as enhancers when cis-linked to the baculovirus 39K early gene promoter. However, a single oligonucleotide containing the palindrome sequence did not bind IE-1 when annealed under conditions conducive to hairpin formation.

Animals↗
PubMed1996

Assessment of the bone mineral density in the lumbar vertebrae of newborns by quantitative computed tomography.

OBJECTIVE: To assess the true mineral density (BMD, in g/cm3) of the lumbar spine in newborns. DESIGN AND PATIENTS: A postmortem analysis of five infants with gestational ages ranging from 35 to 40 weeks, and birth weights from 2765 to 3200 g, was conducted using dual-energy quantitative computed tomography (QCT; Siemens Somatom DR). A 2 or 4 mm thick slice was obtained for each lumbar vertebra from L1 to L4. The density measured in these vertebrae was corrected by reference to a solid phantom (Osteo-CT) measured simultaneously. A three-dimensional image of the spine (Elscint CT Twin), as well as a photomicrograph of histological preparation from L2 vertebra, were also obtained in another term baby for comparison with the CT results. RESULTS AND CONCLUSIONS: In the range of values studied, the vertebral densities were not dependent on birth weight. BMD values measured in L2, L3 and L4 were not significantly different, but were 10% lower than in L1 in four of five infants. The spatial resolution of the QCT protocol used (0.4 mm) did not permit the differentiation of trabecular and cortical bone, and the vertebral bodies appeared very homogeneous and dense, with a mean density value of 210 +/- 30 mg Ca/cm3, which is 2.5 times higher than the mean maximum value found in young normal adults. These preliminary results highlight the potential of QCT in neonatology. Special protocols will, however, need to be developed for in vivo measurements in this particular paediatric field.

Absorptiometry, Photon↗
PubMed1995

An A-DNA triplet code: thermodynamic rules for predicting A- and B-DNA.

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.

Base Sequence↗
PubMed1995

Alternating and non-alternating dG-dC hexanucleotides crystallize as canonical A-DNA.

We have solved the single-crystal X-ray structures of two different hexanucleotides: the alternating sequence d(Gm5CGm5CGC), and the non-alternating sequence d(Gm5CCGGC). Both of these hexamers crystallize readily as A-DNA in the orthorhombic space group C222(1). Although hexanucleotides have been previously crystallized as Z-DNA, and in one case as B-DNA, this is the first time hexanucleotides have been crystallized as A-DNA. Both hexamers adopt a typical A-conformation, which is surprisingly more similar to the structure of A-DNA fibers than to other A-DNA single crystals. The structure of d(Gm5CGm5CGC) was solved to a resolution of 2.1 A (R-factor = 19.6%). This structure has all of the features characteristic of canonical A-DNA, including it's helical repeat (11.2 bp/turn), helical rise (2.6 A/bp), base-pair displacement (-4.7 A), base inclination angle (16.9 degrees), and sugar puckers that are predominantly 3'-endo. The lower resolution, non-alternating structure has similar overall average values for these parameters. We observed several sequence-dependent correlations in these parameters, especially in the d(CG) base step. These steps have lower twist and rise values, coupled with high roll angles as compared to d(GC) steps. The molecular interactions involved in crystal packing and the detailed structure of the bound water in the crystals, however, are similar to those of longer 8 and 10 bp A-DNA crystal structures. Although the structural effect of cytosine methylation on A-DNA appears to be minimal, this modification significantly affects the ability of these sequences to crystallize as A-DNA. In conclusion, we present the A-DNA forming class of hexanucleotides, a new crystallographic system for studying DNA structure at near atomic resolution.

Crystallization↗
PubMed1995

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.

Base Sequence↗
PubMed1995

Inborn errors of metabolism in Singapore.

Between 1992 and 1994 the Paediatric Metabolic Centre screened 403 patients for inherited metabolic disease. Seventeen patients were diagnosed, including 7 with organic acidurias, 5 with mucopolysaccharidoses, 2 with amino acid disorders, 2 with carbohydrate disorders and 1 with hyperlipidaemia. The ethnic distribution of disorders in Singapore is compared with incidences reported by others in this part of the world. It is likely that further studies will indicate which diagnostic or screening tests would best serve this community.

Adolescent↗
PubMed1995

Male homosexual identity in Hong Kong: a social construction.

This research is to explore the social and psychological forces that influence the identity of a man who has come to describe himself as a "homosexual." An attempt is made to understand the emergence of male homosexual identities in Hong Kong--which is predominantly a Chinese community under western influence for more than one century. The results suggest that male homosexual identity arises not so much from homosexual behavior per se but from the stigma and heterosexist beliefs that encompassed it. The acquisition of homosexual identity is largely a response to the cultural definitions of marriage and family, gender and sex roles, as well as a way to handle a culturally induced set of difficulties of getting access to emotional and sexual fulfillment in an environment that prohibits it.

Adult↗
PubMed1994

The non-B-DNA structure of d(CA/TG)n does not differ from that of Z-DNA.

A number of recent studies have shown that simple repetitive d(CA/TG) dinucleotide sequences adopt a left-handed non-B-DNA structure under negative superhelical stress. The pattern of chemical reactivities and the helical parameters observed for these sequences differ significantly from those of standard Z-DNA. In this study, the data for two naturally occurring d(CA/TG)n sequences are reevaluated by a statistical mechanics treatment of the B- to Z-DNA transition. The behavior of these sequences under negative superhelical stress is accurately simulated by this model, including the multiple and discrete transitions observed for the rat prolactin promoter. Furthermore, the average helical twist for the left-handed structure of d(CA/TG)n deviates < 2% from that expected for standard Z-DNA. Finally, the predicted distribution of the junctions between B- and Z-DNA are shown to account for differences observed in the patterns of chemical reactivity of d(CA/TG)n and d(CG)n. Thus, no new left-handed structure that differs from Z-DNA is needed to describe the supercoil-induced conformation in d(CA/TG)n sequences.

Animals↗
PubMed1994

Modeling two dimensions of patient satisfaction: a panel study.

Measurement of patient satisfaction is an essential part of health outcome assessment. The purpose of this study is (a) to compare the stability of an aggregate summary measure and two separate summary measures of patient satisfaction; and, (b) to examine causal effects between two separate summary measures at two points in time. Data were collected from 1,451 Medicare HMO beneficiaries. In conclusion, (a) separate summary measures are recommended for constructing the measure of patient satisfaction and (b) moderate causal relationships and cross-lagged effects between the patient satisfaction measures of qualify of care and access to care were found.

Aged↗