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E Hsu

Publications and source records attributed to E Hsu.

At least 55 records · Page 3Linked to original sources
PubMed1994

Strategies to suppress aggregation of recombinant keratinocyte growth factor during liquid formulation development.

Recombinant human keratinocyte growth factor (rhKGF) is a fairly unstable protein, posing a challenging problem for long-term storage. During storage, the protein unfolds at relatively low temperatures and the unfolded proteins aggregate rapidly, leading to the formation of large visible precipitates. Thermal unfolding of rhKGF displays a similar pattern, i.e., unfolding is followed immediately by aggregation as the temperature is increased. As the unfolding and aggregation (precipitation) of rhKGF limit the storage life of the protein, a search for stabilizers to suppress rhKGF unfolding and aggregation has been done by examining the effects of excipients on thermal melting temperature and on the rate of protein aggregation during storage. Sulfated polysaccharides and citrate are found to be effective in increasing the melting temperature of rhKGF or preventing its aggregation. In particular, 0.5% (w/v) heparin and high molecular weight dextran sulfate, and 0.5 M citrate are highly effective, decreasing the rates of rhKGF aggregation by about 50-fold. Other negatively charged small ions, such as phosphate, also have moderate stabilizing effects on rhKGF. A mechanistic study of the aggregation pathway of rhKGF has led to a better understanding of the stabilizing effects of these molecules. Molecules which enhance rhKGF conformational stability are capable of effectively suppressing rhKGF aggregation.

Animals↗
PubMed1994

Relaxation-time and diffusion NMR microscopy of single neurons.

Relaxation-time and diffusion-weighted NMR micrographs have been obtained for single neurons isolated from Aplysia californica. These images allow the nucleus and cytoplasm to be clearly differentiated, in contrast to proton spin-density images, which appear relatively homogenous. Images of the spatial distribution of T1 and T2 relaxivities and the diffusion coefficient (D), as well as average values for T1, T2, and D in the cytoplasm and nucleus, were calculated from sets of appropriately weighted images. In all cases, water in the nucleus had relaxation and diffusion properties markedly differing from those of cytoplasmic water, which in turn had properties which were distinct from those of free water. Additionally, the cytoplasmic T2 was observed to triple following cell death, which is attributed to cytoplasmic dilution as water enters the cell. The work presented represents the first effort at a consistent exploration of the spatial distribution of NMR characteristics of water within intact single cells. These studies have implications both for modeling the NMR characteristics of water in neuronal tissues based on an understanding of the characteristics of water in different cell compartments and for understanding water/macromolecule interactions within cells. NMR microscopy studies such as these may help form a foundation for understanding and interpreting NMR characteristics measured from large assemblies of cells, i.e., spectroscopy and imaging of living tissues.

Animals↗
PubMed1994

The variation in immunoglobulin heavy chain constant regions in evolution.

The sequences of immunoglobulin (Ig) heavy chain constant (C) region genes do not appear to be well conserved in evolution. The nature of C region change was examined by comparing VH and CH segment variability in the shark, which has numerous C mu as well as VH genes. The sequence diversity among VH was found to be similar to that among C mu 1, suggesting that one Ig segment does not change faster than the other. Although the frequencies of nucleotide substitution were similar, changes in the form of insertions or deletions in loop segments occurred more often in C; the resulting loss of sequence continuity in C exons would make it more difficult to recover C region sequences by crosshybridization. Thus the C regions would seem to be less well conserved than they are. The C region sequences isolated from different animals are described, and sequence and structure are discussed with respect to functions found in mammalian Ig. It is suggested that, although fish, amphibians and reptiles have fewer Ig classes than mammals, heterogeneous gene products are in fact produced and may mediate different effector functions. A theory based on C region selection is presented to explain antibody maturation in animals with a multicluster Ig gene organization.

Amino Acid Sequence↗
PubMed1993

Studies of a sperm/placenta cross-reacting antigen, STX-10.

A monoclonal antibody, HSA-10 initially produced against acrosome-reacted human sperm was also shown to cross-react with human placenta/trophoblast. Transmission electron microscopy, as well as indirect immunofluorescent assay, demonstrated that HSA-10 was found to react with antigen on the inner acrosome of human sperm. The cognate antigen, designated as STX-10, was found to exist as an aggregate in the native form when analyzed by Sephacryl S-300 gel filtration chromatography. When purified by HSA-10-immunoaffinity chromatography from human placenta extract, STX-10 was found to be predominantly a group of glycoproteins with a subunit molecular mass in the range of 75 +/- 5 kDa, whereas an additional group of three proteins with subunit molecular mass less than 20 kDa were copurified from human sperm extract. A sandwich enzyme immunoassay was designed to quantitatively determine the immunoactivity of STX-10 in solution, using HSA-10 monoclonal antibody for coating and for signal detection via enzyme conjugation. Based on this assay, it was found that STX-10 could be detected only in human sperm and placenta extract, but not in any other human somatic tissues, such as serum, brain, heart, muscle, kidney and liver. The immunoactivity of STX-10 was found to be sensitive to proteolytic digestion, low pH, in the presence of reducing agent, but resistant to treatment with sodium periodate. This observation suggests that HSA-10 specific epitope is a peptide in nature and not a carbohydrate moiety. Results of antifertility studies revealed that HSA-10 significantly inhibited human sperm penetration to zona-free hamster ova. Thus, the results of this study are consistent with those of WHO Workshop evaluations that seem to suggest that STX-10 is a highly gamete-specific antigen localized on the inner acrosome of human sperm and in human trophoblast/placenta. Therefore, it may play an important role during human fertilization and embryo development.

Animals↗
PubMed1993

Molecular identity of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.

The molecular identity of mouse sperm acrosome antigen recognized by HS-63 monoclonal antibody was analyzed by various biochemical, immunological and molecular biological methods. When its cognate antigen, MSA-63 was isolated from mouse testis by immunoaffinity chromatography, a group of protein spots with wide range of molecular sizes and isoelectric points were identified. Through previous studies, it was established that most of these protein spots were actin-like molecules co-purified with MSA-63 protein from mouse testis. To analyze the molecular size heterogeneity of the isolated MSA-63 proteins, rabbit antisera against a computer-predicted antigenic synthetic peptide (amino acid residue No. 160-171) and a recombinant glutathione S-transferase (GST) fusion protein (GST-63) were raised. These two antisera and those raised against the isolated MSA-63 protein were used as the probes in comparative Western blot assay, indirect immunofluorescent assay and enzyme-linked immunosorbent assay (ELISA). Using ELISA, antisera against GST-63 and computer-predicted antigenic synthetic peptides were shown to cross-react with affinity-isolated MSA-63 protein coated on microwells. However, little immunological cross-reactivity was observed between GST-63 fusion protein and the synthetic peptide. By using a Western blot assay, two major protein bands of 22 and 32 kDa, respectively were commonly detected on mouse testis homogenate strips by both anti-MSA-63 and anti-GST-63. In addition, anti-MSA-63 also recognized several protein bands with molecular masses greater than 35 kDa. The results of this study suggested that the molecular heterogeneity of MSA-63 protein isolated from mouse testis and sperm, is due to a series of post-translational modifications on a single gene product. These modifications may include glycosylations, proteolytic digestions and tight non-covalent associations with other testicular cytoskeletal proteins, such as actins.

Acrosome↗
PubMed1993

IgH diversity in an individual with only one million B lymphocytes.

Immunoglobulin sequences from an individual Xenopus laevis froglet were analyzed for combinatorial and junctional diversity. In an animal with about 10(6) B lymphocytes, at least 26 out of the 56 VH1 genes available in a diploid genome were expressed, as were all JH segments. Junctional diversity was similar to that observed in Xenopus tadpole sequences, that is, little or no N diversification was found and the recombination site sometimes occurred in a region of V/D or D/J homology. The froglet IgH diversity is further restricted by the elimination of D-gene participation through direct V to J joining. Of the six complementary-determining regions (CDR) contributing to the structure of the antigen-combining site, CDR3 is the most variable in sequence and structure. Froglet IgH CDR3 are restricted to both aspects. Compared to IgH sequences isolated from a 5-month-old adult, froglet CDR3 were, on the average, two codons shorter; overall, 58% of the froglet Ig sequences isolated carried CDR3 of < or = 7 codons, compared to 30% of the adult sequences. In addition to being shorter, the tadpole/froglet CDR3 are less variable in sequence, as the absence of N regions also results in the V/D and D/J junctions being derived from germline elements. We therefore suggest that latent anti-adult specificities are not eliminated in situ, in the tadpole, but rather that such germline gene segments, singly or in their combinations thereof, that can potentially react to adult self-epitopes after metamorphosis have been counterselected during the course of evolution.

Animals↗
PubMed1992

What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4- to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.

Amino Acid Sequence↗
PubMed1992

Changes in the amphibian antibody repertoire are correlated with metamorphosis and not with age or size.

Tadpole and adult Xenopus, manipulated to be of comparable size, exhibited stage-specific antibody expression. The production of adult-type higher-affinity anti-DNP antibodies proved to be independent of the age and size of the individual and is concomitant with the completion of metamorphosis. The appearance of new antibody specificities at such a time suggests that their expression occurs with the cell turnover and renewal during a period of morphological changes.

Animals↗
PubMed1992

Intravascular ultrasound (IVUS) in patients with peripheral arterial occlusive disease (PAOD).

The new method of intravascular ultrasound (IVUS) permits an exact documentation of the pathomorphology of diseased arteries in a two-dimensional cross section image. Exact quantification of the vessel transverse diameters is achiveable. The different ultrasonic properties of fat, connective tissue and calcified tissue allow evaluation of intimal plaque composition. Thrombotic arterial occlusions can be detected. First experiences with IVUS prove, that this method is clearly superior to contrast arteriography and transcutaneous ultrasound, in terms of characterization and quantification of the extent of vessel wall disorders. Distinct structures, such as dissecting membranes or intimal flaps, which may escape arteriographic detection, are clearly visualized by IVUS. False adjustment of the physical parameters of the ultrasonic beam and non coaxial orientation of the ultrasound catheter within the vessel lead to false quantification of the transverse vessel diameters and to over- or underestimation of the echodensity and as a consequence of the intimal plaque composition. Simultaneous imaging of neighbouring arteries, veins or arterial branches allow longitudinal orientation within the vessel. Exact orientation however is only provided by simultaneous fluoroscopy. The present study demonstrates the value of IVUS as a guiding method of transcutaneous interventional procedures. The advantage could be an optimal selection of the recanalizing technique, appropriate to the arterial lesion. The result of interventional therapy can be documented immediately. Furthermore specific injuries of the vessel wall, leading to additional interventions and influencing short and long-term prognosis, can be imaged with high accuracy.

Angioplasty, Balloon↗
PubMed1992

Neuroblastoma and fetal exposure to phenytoin in a child without dysmorphic features.

Despite the fact that the teratogenic effects of phenytoin have been suggested in several case reports, the evidence for a possible oncogenic potential of phenytoin has not been widely recognized. Recently, neuroblastoma as well as other neuroectodermal and non-ectodermal tumors has been seen in several children exposed to phenytoin prenatally. Previous cases have been almost uniformly associated with the features of "fetal hydantoin syndrome" and none have been developmentally normal. We report a developmentally-normal boy of 21/2 years with an abdominal neuroblastoma whose mother had been on phenytoin (as well as carbamazepine) throughout gestation. We review the various neoplasms which have been reported in the offspring of mothers receiving phenytoin.

Abnormalities, Drug-Induced↗
PubMed1991

Physical map and genetic early region of the T7-related coliphage, BA14.

The chromosome of the bacterial virus, BA14, a member of the T7 lytic coliphage group, was characterized by direct measurement of its length and construction of a restriction map. The chromosome (39.6 kb) is essentially the same size as T7 (39.9 kb), is devoid of a large number of restriction sites expected for a DNA of this size, and moreover, lacks modification sites for the Escherichia coli Dam and Dcm methyltransferases. The BA14 early region was assigned by testing the ability of specific chromosomal restriction fragments to direct RNA synthesis by E. coli RNA polymerase, and analysis of in vitro RNase III cleavage products of the transcripts. The data support and extend the previous assertion that BA14 is a representative of a distinct T7 subgroup, and limited nucleotide sequence analysis of the BA14 DNA ligase-encoding gene suggests a closer relationship of BA14 to T7 than to T3 phage, another member of the T7 group.

DNA, Viral↗
PubMed1991

Light chain heterogeneity in the amphibian Xenopus.

Three subpopulations of light chains in Xenopus can be distinguished by monoclonal antibodies as well as by electrophoretic mobility on SDS-PAGE, peptide map and cell surface distribution. Analysis of these proteins from LPS-stimulated lymphocytes culture supernatants by two-dimensional gel electrophoresis showed a heterogeneity comparable to that observed for mouse kappa light chains. However, evidence from the selective expression of light chain subpopulations, as well as highly restricted light chain representation in anti-DNP antibodies, supports earlier findings that an antibody response in Xenopus is greatly limited in heterogeneity.

Animals↗
PubMed1991

Reliability and validity of the objective structured clinical examination in paediatrics.

The assessment of clinical competence has traditionally been carried out through standard evaluations such as multiple choice question and bedside oral examinations. The attributes which constitute clinical competence are multidimensional, and we have modified the objective structured clinical examination (OSCE) to measure these various competencies. We have evaluated the validity and reliability of the OSCE in a paediatric clinical clerkship. We divided the examination into the four components of competence (clinical skills, problem-solving, knowledge, and patient management) and evaluated the performance of 77 fourth-year medical students. The skill and content domains of the OSCE were carefully defined, agreed upon, sampled and reproduced. This qualitative evaluation of the examination was both adequate and appropriate. We achieved both acceptable interstation and intertask reliability. When correlated with concurrent methods of evaluation we found the OSCE to be an accurate measure of paediatric knowledge and patient management skills. The OSCE did not correlate, however, with traditional measures of clinical skills including history-taking and physical examination. Our OSCE, as outlined, offers an objective means of identifying weaknesses and strengths in specific areas of clinical competence and is therefore an important addition to the traditional tools of evaluation.

Clinical Clerkship↗
PubMed1991

[Effect of intra-arterial and intravenous PGE1 infusions on transcutaneous oxygen pressure in patients with critical ischemia of the extremities].

In a randomized cross-over study 20 patients with peripheral arterial occlusive disease stage IV according to Fontaine received one i.a. infusion of 10 micrograms PGE1 in 25 ml saline over 30 min and one i.v. infusion of 40 micrograms PGE1 in 125 ml saline over 60 min. Transcutaneous PO2 was measured continuously (44 degrees C) on the forefoot and dorsum of the foot of the affected leg as well as on the forefoot of the contralateral limb. During i.a. infusion tcPO2 decreased by 73% on the forefoot combined with pain in the infused leg in 8 patients, whereas i.v. infusion induced a 83.1% increase in tcPO2. All in all i.v. PGE1 infusion resulted in a more pronounced increase of tcPO2 without causing side effects.

Alprostadil↗