Biomedical subjects
Publications and source records attributed to T Lee.
A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland lambda gt11 cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.
We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.
Non-isotopic immunoassays for human tumor necrosis factor alpha (TNF alpha) and human interleukin-6 (IL-6) were established by employing the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) system based on the time-resolved fluoroimmunoassay technique with europium-labeled antibody. Compared to enzyme-linked immunosorbent assays and bioassays, the sensitivity and range of measurement were significantly increased by applying the DELFIA systems to TNF alpha and IL-6. TNF alpha was measurable from 100 fg/ml to 10 ng/ml with the TNF alpha-DELFIA and IL-6 was measurable from 100 fg/ml to 1 ng/ml with the IL-6-DELFIA.
Rats were pretreated with 40 mg/kg/day cocaine for 14 days by either subcutaneous injections or osmotic minipumps. Rats were then withdrawn from the pretreatment regime for 1 or 7 days and given a 20-mg/kg IP cocaine challenge (day 1) or a 0-, 10-, 20-, or 40-mg/kg IP cocaine challenge (day 7). The results indicate that rats receiving intermittent, daily injections exhibited sensitization to the behavioral effects of a cocaine challenge on days 1 and 7 of withdrawal. In contrast, rats receiving continuous cocaine exhibited tolerance to the behavioral effects of a cocaine challenge on days 1 and 7 of withdrawal. The present results support and extend previous research that indicates that the route and temporal pattern of administration influences the effects of chronic cocaine. Furthermore, the present results indicate that the continuous infusion paradigm may represent an alternative animal model of some aspects of high-dose cocaine abuse, as compared to the typical procedure of single, or multiple, daily cocaine injections.
HYPOTHESIS: Pediatric endotracheal (ET) tubes can be accurately selected based on body length using a specialized emergency tape. POPULATION: Derivation set: Two hundred five children undergoing elective surgery. Validation set: Two hundred thirteen children undergoing elective surgery. Each child served as his or her own control. METHODS: Derivation phase: Two hundred twenty-one children undergoing ET intubation for elective surgery had their body length and leak pressures measured. The 205 children who had leak pressures between 10 and 40 cm H2O constituted the derivation set. The body length for a given ET tube size was derived from the interquartile range of patient lengths in this derivation set. Sizes for other resuscitative equipment items were chosen by a panel of experts using a modified Delphi technique. This information was placed by length on a color-coded tape. Validation phase: The tape was validated by using it to select ET tube size in another group of 203 children undergoing elective surgery. Criteria for acceptable fit in this group included leak pressure as above and the anesthesiologists' decision to accept the tube size or to retintubate. In the validation phase, length-based ET tube selections were compared with age-based rules: (age + 16)/4, and (age + 18)/4. RESULTS: The tape selected the appropriate ET tube size by leak pressure criterion in 77% of the cases and was within +/- 0.5 mm of the "correct" size 99% of the time. This was significantly better (P less than .005) than two widely used age-based rules, which gave the correct initial size in only 47% and 9% of these cases, and were within +/- 0.5 mm for 86% and 59%. The anesthesiologists chose to continue with the tape-sized tube rather than to reintubate in 89% of cases. CONCLUSION: A system for length-based selection of emergency equipment represents a significant adjunct to emergency physicians and paramedics who must deal with critically ill children. Length-based ET tube selection is clearly superior to age-based rules, which are difficult to remember and require accurate estimation of a patient's age.
We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.
Phosphorylation of p34cdc2 can both positively and negatively regulate its kinase activity. We have mapped two phosphorylation sites in Xenopus p34cdc2 to Thr-14 and Tyr-15 within the putative ATP-binding region of p34cdc2. Mutation of these sites to Ala-14 and Phe-15 has no effect on the final histone H1 kinase activity of the cyclin/p34cdc2 complex. Phosphopeptide analysis shows that there is at least one more site of phosphorylation on p34cdc2. When Thr-161 is changed to Ala, two phosphopeptide spots disappear and it is no longer possible to activate the H1 kinase activity of p34cdc2. We suggest that Thr-161 is a third site of phosphorylation, which is required for kinase activity. All three phosphorylations are induced by cyclin. None of the phosphorylations appears to be required for binding to cyclin, as indicated by the ability of the triple mutant, Ala-14, Phe-15, Ala-161, to bind cyclin. The activating phosphorylation that requires Thr- or Ser-161 occurs even in a catalytically inactive K33R mutant of p34cdc2 and hence does not appear to be the result of intramolecular autophosphorylation. We have detected an activity in Xenopus extracts required for activation of p34cdc2 and present evidence that this is a p34cdc2 activating kinase which, in a cyclin-dependent manner, probably directly phosphorylates Thr-161.
Murine sperm bind a proteinase inhibitor of seminal vesicle origin at ejaculation. The inhibitor binds in the acrosomal region of the sperm head and is removed during in utero or in vitro incubation. Adding inhibitor to sperm reduces their ability to bind zonae, while adding the purified inhibitor binding site to cumulus-free, zona-intact oocytes reduces the ability of the oocytes to bind sperm. Immuno-aggregation of the inhibitor binding site results in exocytosis of the acrosome. These observations suggest that the inhibitor binding site may participate in zona binding and the acrosome reaction. If the inhibitor binding site binds both the zona and the seminal inhibitor, then these components should compete with each other for that site on the sperm. We show that purified seminal inhibitor, as well as other proteinase inhibitors, block zona-induced acrosome reactions. Likewise, zona glycopeptides block inhibitor/anti-inhibitor-induced acrosome reactions in a concentration-dependent fashion. The inhibitor/anti-inhibitor-induced acrosome reaction is sensitive to pertussis toxin and proteinase inhibitor and thus is similar to zona-induced reactions. These findings support the suggestion that the trypsin inhibitor binding site on the head of the sperm functions to insure sperm-zona binding and induction of the acrosome reaction.
We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity.
Roentgenographic determination of total lung capacity (TLC) in children, rarely mentioned in the past, differs from the determination of TLC using helium dilution method in that it does not need the measurement of functional residual capacity. Therefore, it is useful for hospitals where pulmonary function testing (PFT) is not available. In this study, 87 out of 125 normal children completed both chest radiographs and PFT. Ellipsoid method which divides the lung field into several sections was used to calculate TLC from chest radiographs. The correlation coefficient of TLC measured by both chest radiographs and PFT was 0.7680, and paired Student's t-test of the mean values of the two groups showed P value greater than 0.05. Among the variables of height, weight, age and body surface area, height alone was able to predict the normal reference value of TLC. Line of regression between TLC and height was expressed as "TLC = 52.776 x Height- 4404.998", and correlation coefficient was 0.8013. Intersubject reproducibility was also verified. It is concluded that chest radiographs can be used to determine TLC in children.
To evaluate the clinical desired accuracy of the left ventricular ejection fractions (EFs) calculated by magnetic resonance imaging (MRI). Within one to three days post coronary angiography and left ventriculogram, twenty-five adults were studied by MR imaging. They had nuclear medicine studies for left ventricular ejection fractions as well. EKG-gated spin-echo 30-msec echo-delay images were obtained in end systole and end diastole in a plane parallel to the ventricular septum. Analysis of ventricular volumes and ejection fractions were performed using the area-length method. The EFs calculation by gated MRI were compared with that obtained by angiocardiography and nuclear medicine studies. The linear regression line obtained for ejection fraction was y = 0.858x +6.813, r = 0.753, p = 0.009; y' = 1.567x'-24.692, r' = 0.783, p' = 0.002. The above figure indicate a reasonable correlation among these three methods.
This novel, competitive immunoassay simultaneously detects seven drugs of abuse in urine. A urine sample is placed in contact with lyophilized reagents, the reaction mixture is allowed to come to equilibrium (10 min), and then the whole mixture is applied to a solid phase that contains various immobilized antibodies in discrete drug-class-specific zones. After a washing step, the operator visually examines each zone for the presence of a red bar. The method incorporates present threshold concentrations that are independent for each drug. In the absence of drug or in the presence of drug in quantities less than the threshold concentration, no colored bar is visible. Samples containing drug(s) at or above the threshold concentration cause a red bar to appear for the appropriate drug(s). Positive and negative procedural control zones are incorporated into each determination. The performance of the assay methodology matches that of instrumented immunoassay systems.
Hepatitis B vaccine has been recommended for high-risk individuals in the United States for more than a decade. This targeted strategy, however, has failed to control hepatitis B virus (HBV) infection. Universal immunization is being considered as an alternative approach, in particular the inclusion of hepatitis B vaccine with routine childhood vaccinations. Data presented herein demonstrate a high degree of efficacy for hepatitis vaccine with hepatitis B immune globulin in preventing perinatal HBV infection in newborns. Immune response to vaccine was dependent in part on the dose administered, with some enhancement of response if the infant was older at the time of initial injection or if the booster dose was given later. Long-term follow-up showed persistence of vaccine-induced antibody for 5 to 10 years in 90% of immunized infants and adults. Only 3% to 5% of these high-risk individuals had serologic evidence of an HBV infection. None of the infections had been symptomatic and none resulted in a chronic HBV carrier state. Thus, immune responses and efficacy of hepatitis B vaccine in infants were excellent, and immunity and protection against clinically significant HBV infection persisted for at least 5 to 10 years, features essential to success of a program of universal childhood immunization against HBV.
A 49-year-old housewife had suffered from classical seropositive rheumatoid arthritis for 24 years. The shoulders and hands were involved but the most severs pain and deformity were in both elbows and knees. Consequently, she was unable to sit on wheel chair and was confined to a bed. And then, she was admitted to the Chiba Rehabilitation Center in September, 1986 for bilateral total knee replacement. In April 1987, she had an operation upon the right knee and in June, she had an operation upon the left knee. About 6 weeks after surgery, she felt numbness in bilateral hands. The diagnosis was cubital tunnel syndrome. This was confirmed by electromyographic studies. Surgical release was undertaken without delay. The day following operation the patient remarked on the absence of numbness in bilateral hands.
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A study comprising in-depth interviews of sisters in all 15 rural clinics in the Mhala district of Gazankulu showed them to experience major problems with support systems: radiophone communication was unreliable (and even absent in 3 clinics); transport was inadequate; the cold chain was not well maintained and essential drugs and equipment were absent (only 1 clinic had all the equipment to manage a neonatal resuscitation adequately). There were also serious deficiencies in facilities, lack of running water and electricity posing the most serious problems (resulting in deliveries and suturing of episiotomies by candlelight). Other pressing problems include inadequate waiting space and patient privacy. Staffing at the clinics fell well below what is required, yet the commitment of staff was often inspiring. The state of the clinics must be seen within the constraints of 'homeland' policy and the unequal distribution of the country's health care resources. Despite these limitations the clinic service can be improved. Recommendations are made for this, concentrating on improving support structures rather than on the more costly improvement of facilities.
Micellar electrokinetic chromatography is used to separate dansylated nucleotides, both normal and modified species. The high separation power allows detection of minor components present in less than 1 part per thousand of the major components. Laser-excited fluorescence is used to detect the separated components at the 6 x 10(-18) mol level or 10(-9) M injected material. Combined with high-performance liquid chromatographic enrichment prior to labeling, this technique can be used to assess DNA damage in carcinogenesis studies.
The total nucleotide sequences of the genomes of two Japanese encephalitis virus (JEV) strains, the attenuated vaccine strain SA14-14-2 and its parental virulent strain SA14, were determined by using the molecular cloning technique. The sequence analysis revealed that both virion RNA molecules were 10,976 nucleotides long with 95 and 585 flanking bases at the 5' and 3' untranslated sequences, respectively. A single, long open reading frame spanning 10,296 nucleotides was observed to encode a polyprotein of 3432 amino acid residues. When these sequences were compared with each other, 57 nucleotide substitutions were found to be scattered all over the genome. Of these, 24 resulted in amino acid changes within viral proteins. Structural proteins C and E contain one and eight amino acid changes, respectively. Of the nonstructural proteins, NS1 contains three, NS2a two, NS2b two, NS3 four, NS4a one, NS4b one, and NS5 two amino acid substitutions. The 5'- and 3'-terminal untranslated regions contain one- and two- point mutations, respectively. These data and comparative studies with other JEV strain genomes provide a molecular basis for investigating attenuation mechanisms of JEV.