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Biomedical subjects

B Lee

Publications and source records attributed to B Lee.

At least 235 records · Page 13Linked to original sources
PubMed1997

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain.

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.

Animals↗
PubMed1997

Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation.

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The V(H) and V(L) domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two "base-loops" that normally connect V(H) beta strand 3 to 3b and V(L) beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the V(L) domain (V(L) 1-40) of murine antibody anti-Tac to the C-terminal half of V(H) (V(H) 42-115). We also fused the C terminus of V(H) by a (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain). Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal half of V(L) (V(H) 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin. Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments. Due to the artificially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains. Analysis of antigen binding of anti-Tac pFv, and of the specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity when compared to anti-Tac scFv. Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37 degrees C. This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.

Amino Acid Sequence↗
PubMed1997

The expression of serotonin receptors by cultured rat Schwann cells is a function of their differentiation: correlation with a quiescent myelinating phenotype.

Previously we reported that cultured rat Schwann cells express 5-HT2A receptors. In the present study we compared the serotonin-responsiveness of Schwann cells with their stage of differentiation. Serotonin-responsiveness occurred in cells that expressed a myelinating marker but not a nonmyelinating marker. We also examined cells to see if those responding to 5-HT were selectively proliferating or quiescent. This was accomplished by pulsing cells with bromodeoxyuridine (BrdUrd), screening them for serotonin responses, and then immunolabeling the same cells with an antibody recognizing BrdUrd. No BrdUrd+ cells responded to serotonin, whereas most BrdUrd- cells did respond, indicating that serotonin receptors are expressed in Schwann cells after they become quiescent. Moreover, detection of the responses is enhanced by reducing concentrations of mitogen-containing serum in the culture medium. Collectively, these results suggest that postmitotic Schwann cells express serotonin receptors while differentiating along the myelinating lineage.

Animals↗
PubMed1997

Effect of aortic impedance on preload-afterload mismatch in canine hearts in situ.

The aim was to examine the influence of the independent increase of aortic wall characteristics from vascular resistance on global left ventricular (LV) function in intact hearts. In 11 anesthetized dogs, aortic stiffness was increased by a constriction of the thoracic aorta using a stiff tube, and then, in nine dogs, total systemic resistance (TSR) was increased by an infusion of angiotensin II. During the above procedures, aortic input impedance and LV dimension were measured. Aortic input impedance spectra revealed the steady increase in the moduli at high frequencies by aortic constriction, indicating the increase in aortic stiffness. At the stage of increased aortic characteristic impedance (Zc) (134% of control) without a change in TSR, both LV end-diastolic diameter (LVEDD, 99%, p < 0.01 and stroke volume (SV, 95%, p < 0.01) decreased significantly without a change in LV end-systolic diameter (LVESD). During the increase in TSR produced by angiotensin II (128% of control) without a change in Zc, LVEDD tended to increase and LVESD significantly increased (103%, p < 0.01) associated with a decrease in SV (94%, p < 0.01). LV systolic mean wall stress was increased by angiotensin II (118%, p < 0.01), whereas it was unchanged by aortic constriction. In conclusion, the isolated increase in aortic stiffness without a change in TSR was associated with a reduction in LV preload and SV.

Animals↗
PubMed1997

Disparity modulation sensitivity for narrow-band-filtered stereograms.

Stereo thresholds for 84% correct detection of sinusoidal disparity corrugations depicted by narrow-band-filtered random dot stereograms were determined for surfaces as a function of (i) luminance center spatial frequency and (ii) disparity modulation frequency. In addition, supra-threshold depth matching functions for two amplitudes of peak-to-trough depth were determined using similar stimuli. Disparity thresholds followed a U-shaped function when plotted against luminance centre spatial frequency from 1 to 8 c/deg. The threshold functions for the three highest corrugation frequencies (ranging from 0.25 to 1 c/deg) formed a single family with a similar bandpass shape and a peak sensitivity at ca 4 c/deg. At the lowest frequency of depth modulation (0.125 c/deg) the shape of the luminance spatial frequency threshold function showed a reduced sensitivity to depth modulations when portrayed by patterns with high luminance centre frequencies (8 c/deg). The similarity of the threshold functions reveals luminance and corrugation frequency to be largely independent dimensions. The finding that the functions are not identical provides some evidence to support a weak luminance spatial frequency selectivity in stereoscopic channels tuned to corrugation frequency.

Depth Perception↗
PubMed1997

Stabilization of protein structures.

The technique of protein stabilization has been improving steadily in recent years, but it is only in the past year or two that the stability of some protein molecules has been improved to the level of those from extreme thermophilic organisms. This was achieved by multiple mutations and often by utilizing the knowledge gained from the homologous protein structures from extreme thermophiles.

Animals↗
PubMed1997

Variants of congenital ocular motor apraxia: associations with hydrocephalus, pontocerebellar tumor, and a deficit of vertical saccades.

BACKGROUND: Congenital ocular motor apraxia (COMA) is characterized by the inability to generate volitional horizontal saccadic eye movements in the absence of other focal neurologic abnormalities. SUBJECTS: We report on two children (ages 5 months and 3 years) whose COMA did not adhere to these classic criteria. The children were followed up clinically with serial ocular motor examinations and neuroimaging over a period of 3 years. RESULTS: In the first child horizontal COMA was associated initially with neonatal communicating hydrocephalus. Two and one half years after the first signs of COMA, a fourth ventricle medulloblastoma appeared. The second child, who recovered from a periventricular hemorrhage caused by perinatal asphyxia, manifested vertical COMA and compensatory vertical head thrusts. CONCLUSIONS: COMA may be associated with hydrocephalus, pontocerebellar tumor, and periventricular hemorrhage. These rare variants of COMA emphasize that the eye movement deficits may arise from several locations, cerebral as well as pontocerebellar, in the neuronal pathways generating saccades.

Apraxias↗
PubMed1997

Discrete representations of the protein C alpha chain.

BACKGROUND: When a large number of protein conformations are generated and screened, as in protein structure prediction studies, it is often advantageous to change the conformation in units of four consecutive residues at a time. The internal geometry of a chain of four consecutive C alpha atoms is completely described by means of the three angles theta 1, tau, and theta 2, where tau is the virtual torsion angle defined by the four atoms and theta 1 and theta 2 are the virtual bond angles flanking the torsion angle on either side. In this paper, we examine the quality of the protein structures that can be obtained when they are represented by means of a set of discrete values for these angles (discrete states). RESULTS: Different models were produced by selecting various different discrete states. The performance of these models was tested by rebuilding the C alpha chains of 139 high-resolution nonhomologous protein structures using the build-up procedure of Park and Levitt. We find that the discrete state models introduce distortions at three levels, which can be measured by means of the 'context-free', 'in-context', and the overall root-mean-square deviation of the C alpha coordinates (crms), respectively, and we find that these different levels of distortions are interrelated. As found by Park and Levitt, the overall crms decreases smoothly for most models with the complexity of the model. However, the decrease is significantly faster with our models than observed by Park and Levitt with their models. We also find that it is possible to choose models that perform considerably worse than expected from this smooth dependence on complexity. CONCLUSIONS: Of our models, the most suitable for use in initial protein folding studies appears to be model S8, in which the effective number of states available for a given residue quartet is 6.5. This model builds helices, beta-strands, and coil/loop structures with approximately equal quality and gives the overall crms value of 1.9 A on average with relatively little variation among the different proteins tried.

Models, Chemical↗
PubMed1997

Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by hypoplastic or absent clavicles, large fontanelles, dental anomalies and delayed skeletal development. The phenotype is suggestive of a generalized defect in ossification and is one of the most common skeletal dysplasias not associated with disproportionate stature. To date, no genetic determinants of ossification have been identified. CCD has been mapped to chromosome 6p21, where CBFA1, a gene encoding OSF2/CBFA1, a transcriptional activator of osteoblast differentiation, has been localized. Here, we describe two de novo missense mutations, Met175Arg and Ser191Asn, in the OSF2/CBFA1 gene in two patients with CCD. These two mutations result in substitution of highly conserved amino acids in the DNA-binding domain. DNA-binding studies with the mutant polypeptides show that these amino acid substitutions abolish the DNA-binding ability of OSF2/CBFA1 to its known target sequence. Concurrent studies show that heterozygous nonsense mutations in OSF2/CBFA1 also result in CCD, while mice homozygous for the osf2/cbfa1 mull allele exhibit a more severe lethal phenotype. Thus, these results together suggest that CCD is produced by haploinsufficiency of OSF2/CBFA1 and provide direct genetic evidence that the phenotype is secondary to an alteration of osteoblast differentiation.

Amino Acid Sequence↗
PubMed1997

Role of T cells in the pathogenesis of autoimmune lacrimal gland disease in MRL/Mp-lpr/lpr mice.

PURPOSE: MRL/Mp-lpr/lpr mice (MRL/lpr) spontaneously develop an autoimmune disease, including lacrimal gland lesions, which are a model for Sjögren's syndrome. Target organ lesions in MRL/lpr mice are composed largely of CD4+ T cells, and treatment with monoclonal antibodies (mAb) against CD4 improves in the systemic autoimmune disease but not the lacrimal gland inflammation. In anti-CD4 mAb-treated MRL/lpr mice, the lacrimal gland lesions are composed largely of CD8+ T cells. The effects of depletion of: (1) all T cells; (2) both CD4+ and CD8+ T cells, and (3) only CD8+ T cells on the lacrimal gland disease were investigated. METHODS: MRL/lpr mice underwent neonatal thymectomy and were treated with weekly injections of 6 mg of anti-Thy 1 mAb from age one week until sacrifice at age five months. Control nonthymectomized mice underwent similar treatment with either saline or normal rat immunoglobulin (rIg) injections. In a second experiment, MRL/lpr mice were treated with weekly injections of either: (1) 2 mg anti-CD4 mAb and 5 mg anti-CD8; or (2) 5 mg anti-CD8 alone. Control mice underwent similar treatment with either saline or rIg injections. RESULTS: Combined treatment with neonatal thymectomy and anti-Thy 1 mAb was effective in reducing the lacrimal gland disease in both frequency (50% > or = grade 3 vs. 100% in controls, P < 0.002) and extent (median 0% of lacrimal gland area involved by inflammation vs. 14.8% in controls; P = 0.01). Combined anti-CD4 and anti-CD8 therapy also was effective in reducing the lacrimal gland disease in terms of frequency (25% grade 3 vs. 93% in controls; P = 0.002) and extent (median 0% of lacrimal gland involved by inflammation vs. 12.9% in controls; P = 0.0005). Treatment with anti-CD8 mAb therapy alone was ineffective. The systemic autoimmune disease was also improved by T cell depletion and by combined anti-CD4 and anti-CD8 mAb therapy but not by anti-CD8 mAb therapy alone. CONCLUSIONS: Suppression of both CD4+ and CD8+ T cells is required to suppress lacrimal gland inflammation in MRL/lpr mice.

Animals↗
PubMed1997

Pediatric hand injuries due to exercise bicycles.

METHODS: This is a 5-year retrospective review of hand injuries caused by exercise bicycles among 34 children. RESULTS: Mean age was 3 years and 5 months. Accidents were caused most commonly by impingement of the fingers between the chain and sprocket wheel (77%), but injuries caused by spinning wheel spokes (23%) were generally more severe. Injuries were usually of the avulsion type and ranged in severity from lacerations (43%) to fractures (19%) to amputations (38%). The central digits (87%) were involved more frequently than the border digits (13%). Replantation was possible for 33% of the amputated digits, with 50% of the fingers surviving. CONCLUSION: The avulsive nature of these injuries should be taken into consideration when attempting replantation. Increased community awareness and equipment safety modifications are needed to eliminate this preventable problem.

Accidents, Home↗
PubMed1997

Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.

Animals↗
PubMed1997

Atrial natriuretic peptide and cyclic nucleotides affect glucose-induced Ca2+ responses in single pancreatic islet beta-cells: correlation with (Ca[2+] + Mg2+)-ATPase activity.

Glucose stimulation of pancreatic islets is characterized by an initial decline in intracellular Ca2+ concentration ([Ca2+]i) (phase 0), followed by an increase in peak [Ca2+]i (phase 1). The effect of atrial natriuretic peptide (ANP) and cyclic nucleotides on the glucose-induced phase 0 [Ca2+]i was investigated by Fura-2 fluorescent imaging in single beta-cells from isolated islets of rats maintained at 1.67 mmol/l glucose. ANP (1 nmol/l to 1 micromol/l) inhibited the glucose (8.2 mmol/l)-induced phase 0 [Ca2+], in a concentration-dependent manner. Forskolin, 8-bromo-cyclic AMP (8BrcAMP), and 8-bromo-cyclic guanosine monophosphate (8BrcGMP) also inhibited the glucose-induced phase 0 [Ca2+]i. The Ca2+ channel blocker, D 600, prevented the response to 8BrcAMP but not to ANP or 8BrcGMP on phase 0 [Ca2+]i. Thapsigargin (TG) also inhibited phase 0 [Ca2+]i by 90%. ANP, 8BrcGMP, and TG also reduced the time required for glucose to initiate the phase 1 increase in [Ca2+]i, and each of these agents potentiated the effect of glucose on peak [Ca2+]i. Furthermore, sarco(endo)-plasmic reticulum (Ca[2+] + Mg2+)-ATPase (SERCA) activity in RINm5F insulinoma cells was inhibited by 8BrcGMP and TG, but not 8BrcAMP. Thus, ANP and cGMP modulate [Ca2+]i regulation in pancreatic beta-cells perhaps through mechanisms involving changes in SERCA activity and Ca2+ influx.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

CNS innervation of the urinary bladder demonstrated by immunohistochemical study for c-fos and pseudorabies virus.

The aim of the present study is to verify the functional and anatomical neural pathways which innervate the urinary bladder in the central nervous system of the rat. To identify the functional neural pathway, the urinary bladder was stimulated by infusing formalin for 2 h. Then, brain and spinal cord were dissected out and immunohistochemistry was done by using anti-c-fos antibody. Many c-fos immunoreactive (IR) neurons were identified in the telencephalic cortical areas and in several brainstem nuclei, which are known mostly to be related with urinary bladder. In the spinal cord, a number of c-fos IR neurons were found in the lamina I, IIo, dorsal gray commissure, sacral parasympathetic nucleus. To identify the anatomical neural pathway of the urinary bladder, Pseudorabies virus (PRV) was injected into the wall of urinary bladder and was identified with anti-PRV by using immunohistochemistry. Most PRV labeled neurons were found where c-fos IR neurons were identified and few of them were also in the areas where c-fos IR neurons were not found, e.g., prefrontal cortex, agranular insular cortex, and subfornical organ. In the spinal cord, PRV labeled cells were found all over the gray matter. The present study presents morphological evidence demonstrating the supraspinal areas are related with the neural control of the urinary bladder and most functional neural pathway of the urinary bladder is well consistent with the anatomical neural pathway except in some telencephalic cortical areas.

Animals↗
PubMed1997

Hippocampal pathology.

Medial temporal sclerosis of the hippocampus and other lesions in the adjacent temporal lobe that can cause epilepsy are discussed in this article. The technical factors to consider to optimally image the hippocampus and criteria to diagnose medial temporal sclerosis are emphasized.

Atrophy↗
PubMed1997

Mechanisms of bradykinin-induced insulin secretion in clonal beta cell line RINm5F.

We investigated the mechanisms underlying bradykinin (BK)-induced rise in intracellular Ca++ concentration [Ca++]i and insulin secretion using clonal beta cell line RINm5F. Incubation with a range of concentrations of BK increased in concentration-dependent manners both insulin secretion (BK of 10 nM to 10 microM) and [Ca++]i (BK of 100 nM to 100 microM). In Ca++-containing medium, BK (1 microM) induced a biphasic [Ca++]i rise, which was characterized by a Ca++ peak and a sustained Ca++ phase. In the Ca++-free medium, BK failed to increase insulin secretion and induced only a Ca++ peak without the sustained Ca++ phase. Thapsigargin (1 microM), an inhibitor of the Ca++ pump in the endoplasmic reticulum, abolished the Ca++ peak and the sustained phase. Nimodipine (1 microM), a voltage-dependent Ca++ channel blocker, abolished the BK-induced sustained Ca++ phase and inhibited BK-induced insulin release. The BK1 receptor agonist des-Arg9-BK (1 microM) did not change either [Ca++]i or insulin secretion. Both the BK-induced insulin secretion and rise in [Ca++]i were inhibited by a selective BK2 receptor antagonist, HOE 140 (3.3-100 nM), in concentration-dependent manners but were not by a BK1 receptor antagonist des-Arg9,Leu8-BK (1 microM). Pretreatment with pertussis toxin (0.1 microg/ml) did not block the BK-induced insulin secretion or increase in [Ca++]i. U-73122 (4, 6 and 8 microM), a phospholipase C inhibitor, antagonized both the BK-induced insulin secretion and the increase in [Ca++]i in a concentration-dependent and parallel manner. BK increased intracellular concentrations of inositol-1,4,5-trisphosphate (IP3). Neither (p-amylcinnamoyl)anthranilic acid (100 microM), a phospholipase A2 inhibitor, nor N(G)-nitro-L-arginine methylester (100 microM), a nitric oxide synthase inhibitor, inhibited these effects of BK. Taken together, these findings suggested that in beta cells, BK activates BK2 receptors, which, in turn, activate a pertussis toxin-insensitive G protein. The G protein couples to phospholipase C, which promotes the formation of IP3 and diacylglycerol. IP3 releases [Ca++]i from the intracellular Ca++ store, probably the endoplasmic reticulum, which triggers Ca++ influx via voltage-dependent Ca++ channels and thus increases insulin secretion.

Bradykinin↗