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B Lee

Publications and source records attributed to B Lee.

At least 307 records · Page 17Linked to original sources
PubMed1994

Enthalpy-entropy compensation in the thermodynamics of hydrophobicity.

Using Widom's potential distribution theory (J. Chem. Phys. 39 (1963) 2808; J. Phys. Chem. 86 (1982) 869), a general and a special theorems are derived, by means of which one can judge whether a particular sub-process of an overall process will produce compensating changes in enthalpy and entropy. The enthalpy-entropy compensation phenomena that are observed in the transfer process of a hydrophobic molecule from a non-aqueous phase to water are examined in the light of these theorems. It is concluded that most sub-processes involved in the hydrophobic transfer process are compensating except one, that of inserting a cavity corresponding to the solute molecule in the liquid. The reason that this process is non-compensating, and therefore most responsible for the hydrophobicity, is traced to the hard core overlap between solvent and the solute molecules.

Chemical Phenomena↗
PubMed1994

Role of hydrogen bonds in hydrophobicity: the free energy of cavity formation in water models with and without the hydrogen bonds.

The free energies of cavity formation in water with and without hydrogen bonding potential were computed from the results of a set of Monte Carlo simulation calculations on pure liquid TIP4P water model and on the same model but with the electrostatic charges turned off (Lennard-Jones liquid). The free energies of cavity formation in the Lennard-Jones liquids are higher than or approximately equal to those in TIP4P water, depending, respectively, on whether the Lennard-Jones size parameter sigma is set equal to 3.15 A, which is the value of sigma for TIP4P water, or to 2.8 A, which is the commonly assumed value for the oxygen-oxygen distance between two hydrogen-bonded water molecules. This result indicates that changes in the hydrogen-bonded structure of water and/or in the orientational degree of freedom of water are not essential features in the production of the large free energy change upon cavity formation.

Chemical Phenomena↗
PubMed1994

Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv.

Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv immunotoxins as therapeutic and diagnostic agents.

ADP Ribose Transferases↗
PubMed1994

Characterization of freshly dispersed porcine myometrial cells: evidence for voltage-dependent Ca2+ channels and regulatory receptors.

A patch-clamp study of Ca2+ currents and spectrofluorometric detection of the intracellular Ca2+ concentration [Ca2+]i was performed on porcine myometrial cells that had been isolated by collagenase. Isolated myometrial cells were the typical long cylinder shape. The main length and diameter of myometrial cells were 505 +/- 20 and 11 +/- 0.5 microns (n = 40), respectively, in the prepartum period and 265 +/- 22 and 7 +/- 0.4 microns (n = 40), respectively, in the luteal phase. Immunocytochemistry with an antibody against desmin stained 90% of the cells positively, and about 95% of the cells excluded Trypan blue dye. The basal [Ca2+]i of myometrial cells in the luteal phase and the prepartum period was 119 +/- 12 (n = 30) and 154 +/- 31 nmol l-1 (n = 48), respectively. In prepartum myometrial cells, oxytocin (10(-7) mol l-1) and carbachol (10(-4) mol l-1) increased [Ca2+]i in a biphasic pattern, with a sharp peak followed by a plateau. In cells in the luteal phase, adrenaline (10(-7) mol l-1) plus propranolol (10(-6) mol l-1) produced a biphasic increase in [Ca2+]i. However, in the absence of propranolol, the increase in [Ca2+]i by adrenaline was small. Prostaglandin F2 alpha (10(-6) mol l-1) induced a monophasic increase in the [Ca2+]i in cells in the luteal phase. By depolarizing the cells from -30 to +50 mV from a holding potential of -50 mV, Ca2+ currents were evoked with a threshold at -20 mV, reaching a maximum between 10 and 30 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Arginine vasopressin-stimulated insulin secretion and elevation of intracellular Ca++ concentration in rat insulinoma cells: influences of a phospholipase C inhibitor 1-[6-[[17 beta-methoxyestra-1,3,5(10)-trien- 17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) and a phospholipase A2 inhibitor N-(p-amylcinnamoyl)anthranilic acid.

The present study investigated the mechanism by which arginine vasopressin (AVP) increases insulin secretion in rat insulinoma (RINm5F) cells by using a specific phospholipase C (PLC) inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), and a phospholipase A2 (PLA2) inhibitor, N-(p-amylcinnamoyl)anthranilic acid (ACA). AVP (0.1-100 nM) increased insulin secretion and cytosolic free Ca++ concentration ([Ca++]i) dose-dependently. AVP-induced increases in the intracellular concentration of inositol 1,4,5-trisphosphate (IP3) and [Ca++]i were dose-dependently inhibited by U-73122 (2-8 microM). At 8 microM, U-73122 abolished AVP's effect on IP3 and [Ca++]i, but AVP-induced increases in insulin secretion were only reduced by 35%. In contrast, 8 microM U-73122 did not reduce the ionomycin (a Ca++ ionophore, 100 nM)-induced increase in [Ca++]i. The discrepancy between the results of [Ca++]i and insulin secretion in U-73122 experiments is indicative of the multiple signal transduction pathways associated with the activation of AVP receptors, specifically the Ca(++)-independent pathway. The phospholipase A2 inhibitor ACA (100 microM) did not antagonize AVP (10 nM)-induced increases in insulin secretion. These results suggested: 1) U-73122 blocks PLC activities but fails to block other signal transduction pathways that trigger insulin secretion in these cells and 2) AVP increases insulin release from RINm5F cells through both the PLC-mediated Ca(++)-dependent and Ca(++)-independent pathways.

Animals↗
PubMed1994

Direct radioimmunoassay of progesterone in bovine plasma using danazol (17-alpha-2,4-pregnadien-20-yno(2,3-d)isoxazol-17-ol) as a displacing agent.

The majority of progesterone in plasma is bound to cortisol-binding globulin and albumin carrier proteins. In the determination of plasma progesterone concentration by radioimmunoassay (RIA), it is necessary to remove these carrier proteins or displace the hormone from them. In the present study, we have examined the suitability of danazol (17-alpha-2,4-pregnadien-20-yno(2,3-d)isoxazol-17-ol), a synthetic steroid, to displace progesterone from plasma proteins in a direct RIA of bovine plasma. Accordingly, the progesterone content of bovine plasma samples was measured with a RIA using danazol as a displacing agent (direct RIA) and compared with results obtained with a RIA incorporating a preliminary solvent extraction step (extraction RIA). Danazol did not alter the standard curve for progesterone. Sensitivity (ED80) of the direct RIA (9.8 pg/tube) was comparable to that of the extraction RIA (10.3 pg/tube). Results for progesterone assayed in the direct RIA correlated well (r = 0.99) with the results obtained with the extraction RIA. The direct RIA was shown to be accurate; the mean recovery of known amounts of progesterone added to a sample of pooled bovine plasma was 98.5% +/- 3.29 (SEM). The direct RIA intra-assay coefficient of variation (CV) RIA for samples within the low concentration range (0.1-1.0 ng/mL), the medium concentration range (1.0-3.0 ng/mL) and the high concentration range (3.0-6.0 ng/mL) of progesterone were 8.1%, 8.3% and 7.73%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond [termed disulfide-stabilized Fv (dsFV)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that "hypothetically" could be used for disulfide stabilization without changing the structure or affecting antigen binding. This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli. The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies. Furthermore, the finding that B3(dsFv) was much more stable at 37 degrees C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.

ADP Ribose Transferases↗
PubMed1993

Estimation and use of protein backbone angle probabilities.

A procedure is described for estimating the probabilities for the backbone phi-psi angles of a protein molecule from the data base of known protein structures. The procedure is basically an adaptation of a published secondary structure prediction scheme, applied to the phi-psi angle bins rather than to the secondary types. The phi-psi angle probabilities estimated this way include all effects of local sequence and are "context sensitive" in that the probabilities for a given residue type depend on its position along the sequence. These probabilities can be used to predict the three-dimensional structure of short polypeptides that are stabilized mainly by local interactions only and to predict the protein folding initiation sites, with moderate to good success rates in each case. They are also potentially useful for efficient sampling in a Monte Carlo scheme of protein tertiary structure prediction methods.

Amino Acid Sequence↗
PubMed1993

Current methods of estimating severity for occupational injuries and illnesses: data from the 1986 Michigan Comprehensive Compensable Injury and Illness Database.

National and state estimates of the severity of occupational injuries and illnesses (severity = lost work time = missed work days+restricted work days) have come from the annual Survey of Occupational Injuries and Illnesses (Survey) produced by the U.S. Bureau of Labor Statistics. However, we show that the Survey practice of collecting injury information soon after the accident year reduces substantially the accuracy of missed work day estimates, which constitute 85.3% of the Survey lost work time estimate. To develop an independent estimate of missed work days, the research team created the Michigan Comprehensive Compensable Occupational Injury Database (Michigan Database) by linking state files with injury characteristics to files with workers' compensation information for injuries occurring in 1986. The measure of missed work time (days, weeks, or years) is the cumulative duration of compensation from the "date disability commenced," noted on the first payment form, through follow-up to March 1, 1990. Cumulative missed work time has been calculated or estimated for 72,057 injured workers, more than 97% of the 73,609 Michigan workers with compensable occupational injuries in 1986 identified through the close of the study. Our "best" estimate of missed work days, to follow-up, attributable to both fatal and nonfatal compensable occupational injuries and illnesses is 7,518,784, a figure four times that reported for Michigan by the Survey. When insurance industry data on disbursements are also considered, the estimate of missed work days increases to 8,919,079, a figure 4.75 times that reported by the Survey. When insurance data on reserves for future payments are also considered, the estimate of missed work days increases to 16,103,398, a figure 8.58-fold greater than that obtained for Michigan in the Survey. The Michigan data suggest that the national Survey may have failed to identify almost 373 million of 421 million missed work days in the private sector that have resulted, or will result, from 1986 occupational injuries. The present federal/state system for estimating occupational injury severity by measuring lost work days seriously underestimates the magnitude of the problem. The current policy of obtaining incidence and severity data from the same Survey should be reconsidered. We recommend that national estimates of injury severity be obtained from representative states by using state compensation data and that such estimates be used to evaluate current prevention and rehabilitation strategies. The redesigned occupational safety and health Survey (ROSH Survey) should be revised to permit linkage to compensation data.

Accidents, Occupational↗
PubMed1993

The fibrillin-Marfan syndrome connection.

A few years ago no one would have suspected that the well-known disorder of connective tissue, Marfan syndrome, could be caused by mutations in a recently discovered extracellular component, fibrillin. Likewise, nobody would have predicted that fibrillin represents a small family of proteins that are associated with several phenotypically overlapping disorders. The fibrillins are integral constituents of the non-collagenous microfibrils, with an average diameter of 10 nm. These aggregates are distributed in the extracellular matrix of virtually every tissue. Microfibrillar bundles provide the external coating to elastin in elastic fibers, and serve an anchoring function in non-elastic tissues. At higher resolution, individual microfibrils have a "beads-on-a-string" appearance resulting from the head-to-tail polymerization of multiple fibrillin aggregates. Structurally, fibrillin contains a series of repeated sequences homologous to the epidermal growth factor calcium-binding motif. Characterization of fibrillin mutations in Marfan syndrome patients, together with the elucidation of the structure of the fibrillin proteins, have provided new insights, and raised new questions, about the function of the 10 nm microfibrils. For example, it is possible that the fibrillins, in addition to serving a structural function, might also be involved in regulating cellular activities and morphogenetic programs. It is fitting that the long search for the Marfan syndrome gene has brought a novel group of proteins to the forefront of extracellular matrix biology.

Connective Tissue↗
PubMed1993

Conformational preference functions for predicting helices in membrane proteins.

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the alpha-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in alpha-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 alpha-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known alpha-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements.

Animals↗
PubMed1993

Estimation of the maximum change in stability of globular proteins upon mutation of a hydrophobic residue to another of smaller size.

Although the hydrophobic effect is generally considered to be one of the most important forces in stabilizing the folded structure of a globular protein molecule, there is a lack of consensus on the precise magnitude of this effect. The magnitude of the hydrophobic effect is most directly measured by observing the change in stability of a protein molecule when an internal hydrophobic residue is mutated to another of smaller size. Results of such measurements have, however, been confusing because they vary greatly and are generally considerably larger than expected from the transfer free energies of corresponding small molecules. In this article, a thermodynamic argument is presented to show (1) that the variation is mainly due to that in the flexibility of the protein molecule at the site of mutation, (2) that the maximum destabilization occurs when the protein at the site of mutation is rigid, in which case the value of the destabilization is approximately given by the work of cavity formation in water, and (3) that the transfer free energy approximately gives the minimum of the range of variations. The best numerical agreements between the small molecule and the protein systems are obtained when the data from the small molecule system are expressed as the molarity-based standard free energies without other corrections.

Amino Acids↗
PubMed1993

Neuropsychologic effects of stroke in children with sickle cell anemia.

We examined the nature and extent of neuropsychologic impairment in children with sickle cell anemia (SCA) with or without stroke. Twenty-nine children with SCA received cranial magnetic resonance imaging. Strokes were classified into three groups: diffuse cortical stroke (n = 11), anterior stroke (n = 6), or none (n = 12). Children with SCA and 20 age-matched sibling control subjects then received a neurologic examination and a neuropsychologic battery of tests that included motor, verbal, spatial, attentional, and memory measures. Tests of spatial function showed that children with diffuse cortical strokes were impaired, whereas children with anterior lesions had intrusions of irrelevant material during list recall. There were no significant differences between children with stroke and sibling control subjects on motor, verbal, or memory measures. Six children had evidence of stroke on magnetic resonance imaging without any history of a damaging neurologic event. These children had impaired neuropsychologic performance relative to that of sibling control subjects in a pattern similar to that of children with overt stroke. Children with SCA without stroke did not differ from sibling control subjects on any measure. Our results indicate that overt and silent strokes result in lesion-specific neuropsychologic deficits in children with SCA.

Adolescent↗
PubMed1993

Standardization of penile blood flow parameters in normal men using intracavernous prostaglandin E1 and visual sexual stimulation.

The evaluation of vasculogenic impotence by color flow Doppler ultrasound after injection of intracavernous vasoactive agents allows for simultaneous visualization in real time of arterial and venous blood flow. Normal arterial blood flow parameters after prostaglandin E1 injection have yet to be standardized. Our study was initiated to evaluate blood flow parameters in a normal control population after prostaglandin E1 and visual stimulation. A total of 20 healthy male volunteers 45 to 60 years old with histories of normal sexual function was selected. All volunteers were given intracavernous injections of 10 micrograms prostaglandin E1 and received concurrent visual stimulation by means of an erotic video. All patients developed rigid erections with no complications. Using color flow Doppler ultrasound measurements were done before and after prostaglandin E1 injection of right and left superficial and deep cavernous artery diameters, peak blood flow velocities and blood flow volumes. Results (mean plus or minus standard error) showed a significant increase in diameters after prostaglandin E1 in the superficial (20% increase) and deep (70% increase) penile arteries. Blood flow volume increased 3-fold for the superficial penile arteries (from 7.3 +/- 1.4 to 20 +/- 3.5 cc per minute) and 4-fold for the deep cavernous arteries (from 3.8 +/- 1 to 12.5 +/- 1.8 cc per minute). Peak blood flow velocity increased 2-fold (from 22 +/- 3 to 46 +/- 7 cm. per second) for the superficial arteries and 3-fold (from 12.5 +/- 2 to 37 +/- 5 cm. per second) for the deep cavernous arteries. These data suggest control values for normal erectile function in middle-aged men as a 70% increase in deep cavernous artery diameter, a systolic peak blood flow velocity greater than 30 cm. per second and more than 10 cc per minute of blood flow volume. With these standards the clinician may assess, design and follow treatment strategies for vasculogenic impotence.

Alprostadil↗
PubMed1993

Treatment of toxic epidermal necrolysis and Stevens-Johnson syndrome in children.

A retrospective study was carried out on eight consecutively treated children, 2 to 14 years of age, seven with toxic epidermal necrolysis and one with transitional-type Stevens-Johnson syndrome. The body surfaces affected ranged from 40% to 100%. Seven of the patients were taking a sulfonamide or anticonvulsant before the onset of their disease. Complete reepithelialization took an average of 15 days, but newly evolving lesions of the lips and oropharynx continued for approximately 4 more weeks. These lesions took an additional 2 to 4 weeks to heal. Only one of the children died.

Adolescent↗
PubMed1993

Thyromimetic effects of 3,5,3'-triiodothyronine sulfate in hypothyroid rats.

Several parameters of the effects of thyroid hormone were examined in hypothyroid thyroidectomized (Tx) rats treated with T3 sulfate (T3S) or T3 [0.46 (low dose) or 2.3 (high dose) nmol/day for 10 days, ip]. Tx rats showed a marked degree of growth retardation, which improved significantly after treatment with both doses of T3S and T3. The mean serum GH level was markedly reduced in Tx rats, and it improved significantly to similar levels after treatment with the high dose of T3S and the low dose of T3. Type I monodeiodinase (MD) activity was markedly reduced in liver and kidney tissues of Tx rats. It increased significantly in Tx rats treated with the high dose of T3S; the latter values were similar to those observed in Tx rats treated with the low dose of T3. Hepatic and renal type I MD activities increased to supranormal levels in Tx rats treated with the high dose of T3. Cardiac outer ring (5') monodeiodination of 3',5'-diiodothyronine to 3'-monoiodothyronine was also significantly reduced in Tx rats, but it improved significantly only after treatment with the high dose of T3. Type III 5-MD activity was significantly reduced in the cerebral cortex of Tx rats. It was restored to normal in Tx rats treated with the high dose of T3S and both doses of T3. Serum TSH, markedly elevated in Tx rats, was appreciably reduced only in rats treated with the high dose of T3. In another study, significant suppression of serum TSH was observed when Tx rats were treated with T3S (11.5 nmol/day) or T3 (2.3 nmol/day) for 3 days. We conclude that administration of T3S to hypothyroid rats produces thyromimetic effects, with a potency approximately one fifth that of T3.

Animals↗
PubMed1993

A study of the 3,5,3'-triiodothyronine sulfation activity in the adult and the fetal rat.

We have employed a new in vitro assay for study of the T3 sulfation activity in rat tissues. The assay measures by RIA the generation of T3 sulfate (T3S) during incubation of T3 with cytosol of rat tissues as the source of phenol sulfotransferase(s) and 3-phosphoadenosine-5'-phosphosulfate as the sulfate donor. The conversion of T3 to T3S proceeded rapidly for 30 min at 37 C, and the optimal pH of the reaction was 8.0. Heating the cytosol at 44 C for 15 min decreased T3S production to 63% of its value at 37 C. T3 sulfation activity was plentiful in rat liver, brain, and kidney, but little activity was demonstrable in other tissues. The Km and maximum velocity of the hepatic conversion of T3 to T3S were 114 microM and 159 pmol/mg protein.h, respectively. There was a marked inhibition of the conversion of T3 to T3S with salicylamide, 3'-monoiodothyronine, thyronine, and rT3; the IC50 of these inhibitors approximated 15, less than 0.1, 9.5, and 43 microM, respectively. On day 17 of gestation, the T3 to T3S conversion activity was more abundant in fetal skin than in other fetal tissues. However, the activity decreased in fetal skin while it increased in fetal liver, kidney, and brain nearer to term on day 20. Placenta demonstrated lower T3 to T3S conversion activity than several fetal or maternal tissues. There was no effect of hypothyroidism or hyperthyroidism on T3 sulfation activity. We conclude that T3 sulfation activity in the rat is 1) most abundant in liver, kidney, and brain tissues of the adult; 2) inhibited more avidly by 3'-monoiodothyronine than other thyronines; 3) very abundant in fetal skin early in gestation; and 4) little affected by the thyroidal status of the animal.

Animals↗