Biomedical subjects
Publications and source records attributed to H Chu.
Treating two cases of vascular palsative tinnitus with digital subtraction angiographic and superselective embolization was reported in this paper. The etiology pathogenesis, clinical sign and its treatment were also discussed.
Lactoferrin (Mw=78 kDa) is a member of the transferrin family of iron-binding glycoproteins. Previous studies carried out primarily in vitro indicate that the protein has multifunctional properties and may be involved in regulation of iron homeostasis, inhibition of bacterial growth and regulation of immune responses. However, the significance and species specificity of these proposed functions in vivo have not been adequately addressed due to lack of sufficient purified homospecies lactoferrin for analysis in small animal models. We previously reported the successful production of biologically active recombinant human lactoferrin using an Aspergillus expression system. In the present study, we report the production of recombinant murine lactoferrin using a similar expression strategy. Recombinant murine lactoferrin was purified to homogeneity and was similar in size and immunoreactivity to native murine milk lactoferrin. The recombinant protein was correctly processed at its N-terminus and was glycosylated. Interestingly, while both human and murine lactoferrin bind iron in a 2:1 molar ratio, iron bound to recombinant murine lactoferrin was more acid labile than human lactoferrin, demonstrating species-specific variation in the stability of iron-binding to this protein. Finally, the availability of recombinant murine lactoferrin will now facilitate the study of the species specificity of lactoferrin action in a mouse model system.
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes (5-HT2A, 5-HT(2B) and 5-HT(2C)) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT(2C)R) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily.
PURPOSE: The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. METHODS: rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37 degrees C. RESULTS: rhGH PLGA formulations were produced with a low initial burst (< 20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37 degrees C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occurred at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. CONCLUSIONS: rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37 degrees C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.
Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the N-hydroxysuccinimide ester of polyethylene glycol-5000 (PEG5000), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC50 in a cell-based assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG5000, and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.
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Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.
The development of the central nervous system in Drosophila is initiated by the segregation of neuroblasts, the neural progenitors, from the embryonic neuroectoderm. This process is guided by at least two classes of genes: the achaete-scute complex (AS-C) proneural genes and the neurogenic genes. It has been known for some time that loss-of-function mutations in the AS-C result in neural hypoplasia and the first observed defect is failure of segregation of a fraction of neuroblasts. Loss-of-function mutations at the ventral nervous system defective (vnd) locus are known to lead to similar phenotypic defects in early neurogenesis. More recently, the vnd locus has been implicated in the regulation of the proneural AS-C genes and the neurogenic genes of the Enhancer of split complex. In this paper we report the identification of a transcript associated with the vnd locus, the transcript distribution in embryogenesis, which is compatible with the nervous system mutant phenotypes described for this gene, and that the protein product is a member of the NK-2 homeodomain family. We discuss these findings within the framework of early Drosophila neurogenesis and the known phenotypes associated with the vnd locus.
There is a polymorphic tetranucleotide short tandem repeats (STR) sequence in the intron 3 of the phenylalanine hydroxylase (PAH) gene. The polymorphism of this STR sequence in Chinese was analysed on 36 normal individuals and 16 PKU families. After PCR amplification, the DNA fragments were separated on polyacrylamide sequencing gel and visualized by silver-staining. Seven alleles were identified with frequencies of 0.028, 0.042, 0.014, 0.138, 0.542, 0.208 and 0.028 in normal populations, and 0.016, 0.078, 0.047, 0.312, 0.375, 0.156 and 0.016 in PKU families, and PICs of 0.585 and 0.693, respectively. This polymorphic marker is very useful in quick prenatal gene diagnosis in PKU families, and prenatal gene diagnosis was performed by linkage analysis in two pregnancies at risk.
The first bone marrow transplants (BMTs) in human patients were performed after conditioning with total body irradiation (TBI). TBI remains an important part of BMT protocols. The morbidity and mortality of BMT remains significant, but can be decreased by the introduction of optimized TBI regimens. This requires dosimetric control and a detailed analysis and description of the physics of the TBI procedure in every BMT center that utilizes TBI. Recommendations for such procedures are given. Radiobiological models are of help in developing less toxic TBI procedures, but can only be effective after dosimetric control has been obtained and if the influence of other variables on the outcome of BMT are taken into account. Fractionated TBI (fraction size over 3.0 Gy or higher) appears to be more effective and better tolerated than single fraction TBI. Lung shielding is possible during TBI. Smaller organs or organs that cannot be imaged easily are not recommended for shielding. Radiolabeled immunoglobulins are but low molecular weight bone seeking radioisotopes and are not expected to improve the therapeutic ratio of TBI. Other variables in BMT are more difficult to quantify and model than TBI (e.g. high-dose chemotherapy, graft-versus-host disease) and will be more difficult to optimize.
Serotonin2A (5-HT2A) and 5-HT2C receptors share numerous pharmacological properties. Two compounds thought to discriminate between these two receptor subtypes are m-chlorophenypiperazine (mCPP) and m-trifluoromethylphenylpiperazine (TFMPP). These two drugs have been classified as antagonists at 5-HT2A receptors but as agonists at 5-HT2C receptors on the basis of phosphoinositide hydrolysis studies in cerebral cortex and choroid plexus, respectively. To determine more fully the properties of mCPP and TFMPP at 5-HT2A receptors, NIH 3T3 fibroblasts transfected with the 5-HT2A receptor complementary DNA (GF6 cells) were used as a model system of receptor function. These cells express approximately 15-fold higher 5-HT2A receptor density than is found in cerebral cortex. In GF6 cells, mCPP and TFMPP dose-dependently stimulated phosphoinositide hydrolysis with maximal effects less than that of 5-HT. This agonist activity was blocked by 5-HT2A receptor antagonists but not by prior treatment with pertussis toxin. Partial inactivation of 5-HT2A receptors with phenoxybenzamine decreased the maximal effects of mCPP and TFMPP but did not eliminate agonist activity. Thus mCPP and TFMPP are partial agonists at 5-HT2A receptors in GF6 cells, and these agonist properties are retained even under conditions where receptor density is comparable to that of cerebral cortex. Although it has not yet been demonstrated that mCPP and TFMPP are agonists at central 5-HT2A receptors, this possibility should be considered when evaluating in vivo effects of these drugs.
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Animals treated acutely with an adenosine receptor antagonist have elevated plasma renin activity. This observation suggests that endogenous adenosine plays a physiologically significant role in restraining renin release. However, it is unclear whether chronic blockade of adenosine receptors would cause a rise of renin activity since tolerance to adenosine blockade is known to develop quickly. An earlier study partially addressed this question by showing that chronic blockade of adenosine receptors with caffeine exacerbated both the rise of plasma renin activity and the decline of renal function in 2-kidney-1-clip (2K1C) renovascular hypertensive rats. However, that study did not determine whether the difference in renin activity occurred solely as a secondary result of the difference in renal function. The purpose of this study was to reexamine the effect of chronic caffeine consumption on plasma renin activity and angiotensin I levels in animals in another high-renin model, i.e., the low sodium diet. The low sodium diet is devoid of the potential confounding effect of deteriorating renal function associated with the 2K1C renovascular hypertension model. In this study, animals received normal rat chow and drank either 0.1% caffeine water or vehicle for ten days. After ten days, all rats were switched to a low sodium diet for three weeks. Plasma renin activity and plasma angiotensin I levels were measured before, and at 1 and 3 weeks after initiating the low sodium diet. The results of this study show that chronic blockade of adenosine receptors with 0.1% caffeine water increases plasma renin activity and angiotensin I concentration before and throughout the three weeks when animals were on the low sodium diet. The results of this study suggest that the inhibitory role of adenosine on renin release is a general physiological process, rather than a special situation applicable only to the 2K1C model.
Roentgenographic techniques were investigated for imaging orthotopic lung tumours in anaesthetized nude rats endobronchially implanted with human lung cancer cells. A conventional radiographic unit with a dual-screen, double-emulsion film mammographic receptor produced images preferable to those from a mammographic unit because of superior resolution. Typical exposure factors were 300 mA, 29 kVp, and 17 ms at a focus-film distance of 76 cm with a 2.11 by 2.41 mm effective focal spot and inherent filtration of 1.2 mm aluminium. Sensitivity for tumour detection was 0.93 for 59 animals with pathologically proved tumours and 0.96 for 54 animals with tumours larger than 4 mm or 50 mg. For 24 pathologically tumour-free animals, specificity was 1.00. For 55 animals radiographically judged to have tumours, positive predictive value was 1.00. For all 83 animals, accuracy was 0.95. This technique effectively demonstrates orthotopic human lung tumours in nude rats and should be useful for noninvasive monitoring of tumour presence, location, size, and changes in size.
The development of improved animal models for biological and preclinical studies of human lung cancer is important because lung cancer is the leading cause of cancer death in the United States. To determine whether the Rowett nude rat could serve as an orthotopic (organ-specific) model of this disease, nude rats (CR: NIH-RNU), with and without 500 rads of prior gamma-irradiation, were implanted intrabronchially with 10(7) cultured cells from 3 human lung cancer lines. Without irradiation, the NCI-H460 large-cell undifferentiated carcinoma had a 54% take-rate, whereas the NCI-H125 adenosquamous carcinoma and A549 adenocarcinoma had take-rates of 7 and 33%, respectively; irradiation increased the respective take-rates to 100, 83, and 90%. In irradiated rats, tumor age versus weight measurements showed progressive growth for all three tumors, with growth rates in the order: NCI-H460 greater than A549 greater than NCI-H125, requiring approximately 3, 5, and 9 weeks, respectively, for average tumor sizes to exceed 500 mg. The small-cell carcinoma cell line NCI-H345 was implanted only into irradiated rats and resulted in more slowly growing tumors. Histopathological study showed all model tumor types to have histological characteristics consistent with the clinical tumors from which the cell lines were derived. Each tumor type had a different growth pattern, with some of the the A549- and NCI-H125-derived tumors metastasizing to contralateral lung and/or regional lymph nodes. There was no evidence for immunological rejection in irradiated, tumor-bearing rats. Nonirradiated, implanted rats without gross tumor exhibited peribronchiolar mononuclear cell infiltration with or without fibrosis, suggesting prior immunological rejection. The successful orthotopic growth of these 4 human lung cancer cell lines in irradiated nude rats suggests that this model could be useful for biological and preclinical studies of human lung cancer, both in intact rats and via ex vivo perfusion of their tumor-bearing lungs.
The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection of Escherichia coli for complementation of a biosynthetic defect. Cloned fragments containing the gene and the P. asaccharolyticus transcription and translation signals are very highly expressed in E. coli. The nucleotide sequence of the cloned gene was determined. It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases. The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.
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Screening of lens homogenates from the representative species of five major classes of vertebrates was undertaken to search for delta-crystallin with argininosuccinate lyase activity. Purification and biochemical characterization of delta-crystallins from the avian and reptilian species revealed differences in their electrophoretic and kinetic properties in spite of their similar tetrameric structure of about 200 kDa in the native forms. Chicken delta-crystallin, in contrast to those obtained from duck, goose and caiman, is almost devoid of the enzymatic activity. Two-dimensional gel electrophoresis of lens homogenates indicated that in the chicken lens delta-crystallin is composed of a subunit with an isoelectric point of 5.9 and a subunit mass of 50 kDa whereas that of goose lenses possesses heterogeneous subunits with isoelectric points spreading in a range of 5.9 to 6.8. Immunological comparison of inactive and active delta-crystallins from the chicken, duck and caiman lenses established the apparent structural similarity of all delta-crystallins to the authentic enzyme regarding some of common surface epitopes, yet they are not completely identical. Kinetic constants for two of the active delta-crystallins, i.e. those from the duck and goose of the Anatidae family, were also determined and their catalyzed reaction was shown to conform to a random Uni-Bi kinetic mechanism similar to that of the argininosuccinate lyase from the bovine liver.