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Biomedical subjects

B J Lee

Publications and source records attributed to B J Lee.

At least 145 records · Page 8Linked to original sources
PubMed1994

Nuclear magnetic resonance study of the interaction of T4 endonuclease V with DNA.

T4 endonuclease V catalyzes the DNA strand cleavage in the vicinity of a thymine dimer. In order to obtain insight into the specific recognition mechanism of this enzyme with a thymine photodimer within DNA, the conformations of five different DNA duplexes, [sequence: see text] with which the enzyme can interact, were studied by 1H NMR. DNA I, DNA IV, and DNA V do not contain the TT sequence or a thymine dimer and hence, are expected to bind the enzyme only in a nonspecific manner. DNA II includes a single TT sequence which does not form a thymine dimer. Only DNA III is expected to bind specifically to the enzyme through a thymine photodimer. The NMR spectra of these five DNA duplexes in the absence of the enzyme clearly show that the formation of a thymine dimer within the DNA induces only a minor distortion in the structure and that the overall structure of B-type DNA is retained. The photodimer formation is found to cause a large change in chemical shifts at the GC7 base pair, which is located at the 3'-side of the thymine dimer, accompanied by the major conformational change at the thymine dimer site. The effects of T4 endonuclease V binding on these DNA duplexes were also investigated by 1H NMR. The binding of this enzyme to DNA I, DNA IV, and DNA V causes no alteration in chemical shift values of the imino proton resonances, but the binding to DNA II induces a small downfield shift in the imino proton resonance of GC7.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1994

Nasal challenge with allergen upregulates the local expression of vascular endothelial adhesion molecules.

To understand the events involved in selective eosinophil migration into allergic inflammatory sites, we studied the expression of vascular endothelial adhesion molecules in the nasal mucosa. Ten subjects with asymptomatic seasonal allergic rhinitis and 13 nonallergic subjects underwent localized allergen challenge of one inferior turbinate. Twenty-four hours later, biopsy specimens were obtained from the inferior turbinates, bilaterally in the seasonally allergic subjects and unilaterally in the nonallergic control subjects. The specimens were divided, sectioned, and either stained for identification of eosinophils or analyzed immunohistochemically for intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and von Willebrand's factor. Intercellular adhesion molecule-1 expression was observed in all mucosal specimens, with no significant difference among groups. E-selectin showed minimal baseline expression, and low levels were significantly induced on the challenged mucosa of the allergic compared with nonallergic subjects (p < 0.05). VCAM-1 was expressed basally and was significantly upregulated by allergen challenge, compared with the nonchallenged side and nonallergic control subjects (p < 0.05). Submucosal eosinophils increased significantly in allergic subjects 24 hours after antigen challenge, compared with nonallergic control subjects and weakly correlated with VCAM-1 expression (rs = 0.33, p = 0.06). Our results suggest that endothelial activation accompanies allergic inflammation. Furthermore, because the counterligand for VCAM-1, very late activation antigen-4, is present on eosinophils, VCAM-1 upregulation may contribute to the selective recruitment of these cells to the nasal mucosa.

Adult↗
PubMed1994

Clonal analysis and virulence of Australian isolates of Streptococcus suis type 2.

Multilocus enzyme electrophoresis was used to divide 124 Australian isolates of Streptococcus suis type 2 into 17 electrophoretic types (ETs). Isolates in ET 1 were the most frequent cause of disease amongst Western Australian pigs, but isolates of ET 8 were more commonly associated with disease in other Australian states. Multiple isolates from 10 of 19 farms all belonged to the same ET, whilst isolates from the other farms belonged to between 2 and 4 different ETs. Some isolates could be differentiated further by DNA restriction endonuclease analysis, whilst others with the same restriction pattern were located in different, but closely-related ETs. Fourteen isolates were tested for their virulence in mice. Most caused disease if given in high numbers, but isolates in ET 1 were virulent at lower dose rates. This virulent clone also was distinguished by the fact that 80% of isolates produced extracellular factor (EF).

Animals↗
PubMed1994

Blockade of noradrenergic neurotransmission with diethyldithiocarbamic acid decreases the mRNA level of gonadotropin-releasing hormone in the hypothalamus of ovariectomized, steroid-treated prepubertal rats.

We have previously found that progesterone (P) augmented gonadotropin-releasing hormone (GnRH) mRNA levels in the hypothalamus of ovariectomized, estradiol-treated (OVX + E) prepubertal rats. In order to determine whether noradrenergic neurotransmission is involved in the stimulatory effect of P on GnRH gene expression, diethyldithiocarbamic acid (DDC, 500 mg/kg), a dopamine beta-hydroxylase inhibitor was administered i.p. 1 h before P (1 mg) injection into OVX + E treated rats, and the effect of DDC on the P-induced GnRH mRNA levels was examined. A single injection of P into OVX + E primed rats augmented norepinephrine (NE) content, while the administration of DDC effectively blocked the P-induced increase in NE content, along with the increase in dopamine content. Suppression of NE neurotransmission with DDC resulted in a marked decrease in the P-induced GnRH mRNA levels as well as GnRH release in vitro. These results clearly demonstrate that noradrenergic neurotransmission is involved in P-stimulated GnRH gene expression in the rat hypothalamus.

Animals↗
PubMed1994

Preliminary evaluation of transdermal delivery of melatonin in human subjects.

A transdermal delivery device (TDD)1 was applied to four human subjects to investigate whether melatonin (MT) could penetrate through human skin. The TDD (total surface area of 3.80 cm2) was applied to the forearm of each subject. Plasma MT concentrations increased above baseline in approximately 2-4 hours, although steady state was not achieved in the 8-hour study period. Intersubject variation of plasma MT among four subjects was noted. Urinary excretion of 6-sulphatoxymelatonin (6-STMT), a major metabolite of MT in humans, increased as plasma MT concentrations increased. Cumulative amounts of urinary 6-STMT increased over a 6-hour period when the TDD was applied and were three times greater than in controls. The urinary excretion rate of 6-STMT was statistically correlated with plasma MT concentration among subjects (r2 = 0.77). These data suggest that the urinary excretion rate of 6-STMT can be used as an index of MT plasma concentrations in human subjects. Although an intersubject variability in both plasma MT concentration and urinary excretion rate of 6-STMT was noted, it was evident that MT can be delivered transdermally in human subjects.

Administration, Cutaneous↗
PubMed1993

Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and its domain fragments were used to map nucleic acid binding sites within the enzyme. Discrete domain fragments were produced after the digestion of three forms of RT (p66, p66/p51 heterodimer, and p51) with V8 protease or trypsin, and the primary structure of each domain fragment was mapped by both immunoblotting and N-terminal amino acid sequence analysis. These domain fragments represent N-terminal, middle, or C-terminal regions of RT. Using Northwestern or Southwestern blotting assays, the domain fragments were evaluated for nucleic acid binding. In this technique, RT proteins are electroblotted onto the membrane and renatured after SDS-PAGE; the proteins are then probed with the primer analogues 32P-labeled d(T)16 or 32P-labeled tRNA(Lys,3). A V8 protease domain fragment spanning residues 195 to approximately 300 (p12), which was found earlier to be UV cross-linked to the primer in intact RT [Sobol et al. (1991) Biochemistry 30, 10623-10631], showed binding to both nucleic acid probes. We first localized nucleic acid binding in p66 to an N-terminal domain fragment of residues 1 approximately equal to 300. By contrast, a C-terminal domain fragment termed p30(303 approximately equal to 560) did not show nucleic acid binding. To investigate the role of the region just N-terminal to residue 303, an expression vector named pRC-35 encoding residues 273-560 was constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

A partial blockade of catecholaminergic neurotransmission with 6-hydroxydopamine decreases mRNA level of gonadotropin releasing hormone in the male rat hypothalamus.

Central catecholamines (CA) are known to be involved in the regulation of synthesis and secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus. However, no attempt has been yet made to determine whether CA affects GnRH gene expression. To this end, the effect of 6-hydroxydopamine (6-OHDA), a catecholaminergic neurotoxin, on GnRH mRNA level was examined. Hypothalamic tissues obtained from adult male rats were incubated with medium containing 6-OHDA. To ensure the effect of 6-OHDA on CA depleting action, CA levels in media and in postincubation tissues were determined. Increasing concentrations of 6-OHDA resulted in decrease in norepinephrine (NE) and dopamine (DA) contents in a dose dependent manner. Treatment with 6-OHDA (5 x 10(-4) M produced a time-dependent decrease in NE but not DA, when CA levels in media were determined at 30 min intervals during the incubation period. To determine changes in GnRH mRNA level in response to 6-OHDA treatment in vitro, for 2.5 h total cytoplasmic RNA fractions were isolated from postincubation hypothalamic tissues and used for RNA-blot hybridization with 32P-labeled GnRH riboprobe. A blockade of CA neurotransmission with 6-OHDA (5 x 10(-4) M) significantly reduced GnRH mRNA level by half over its control and internal control (actin mRNA) groups. Northern blot analysis revealed that addition of NE (1 x 10(-6) M) reversed the decreased GnRH mRNA level by 6-OHDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The effect of ostial opening on experimental maxillary sinusitis in rabbits.

Obstruction of the natural ostium is known to be one of the major factors in the pathogenesis of maxillary sinusitis. To explore the therapeutic effect of ostial patency, sinusitis was induced in 32 rabbits by obstruction of the natural ostium and inocculation of Streptococcus pneumoniae. They were divided into two groups: the first ("open") group included 16 rabbits with experimentally induced sinusitis, from which bone chips for obstruction of the natural ostium were removed after a period of 5 days, and the second ("closed") group included 16 rabbits with continuous obstruction of the natural ostium. The induction of sinusitis was found to be successful in all cases, when examined after five days. The amount of nasal and sinus secretion as well as histopathological findings of the sinus mucosa were investigated with light microscopy, scanning and transmission electron microscopy, two and four weeks after induction of sinusitis. The findings were significantly improved in the "open" group.

Animals↗
PubMed1992

Four cycles of chemotherapy and regional radiation therapy for clinical early-stage and intermediate-stage Hodgkin's disease.

To achieve a high percentage of durable complete remissions (CR) and prolonged survivals and reduce toxicity in patients with early-stage and intermediate-stage Hodgkin's disease, a randomized trial of four cycles of mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) versus four cycles of thiotepa, bleomycin, and vinblastine (TBV) combined with regional radiation therapy (RT) was conducted. For MOPP and RT, the CR percentage was 98% (60 of 61), and at 5 years, the percentage of patients in CR was 90%, with freedom from progression of 89% and overall survival of 91%. For TBV and RT, the CR percentage was 93% (55 of 59), with a 5-year duration of CR of 83%, freedom from progression of 81%, and overall survival of 91% (P greater than 0.15). The median follow-up was 65 months (range, 7 to 96 months). For 27 patients with clinical Stage IIIA, the CR percentage for MOPP and RT was 75% (12 of 16), with 1 relapse and 4 deaths. For TBV and RT, the CR percentage for clinical Stage IIIA was 73% (8 of 11) with 2 relapses and 2 deaths. Short-term toxicity except for transient leukopenia was less for TBV and RT than for MOPP and RT. Good results are achievable with combined treatment without excessive toxicity.

Adolescent↗
PubMed1992

Regional localization of the selenocysteine tRNA gene (TRSP) on human chromosome 19.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.

Chromosome Banding↗
PubMed1991

Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding.

Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component. Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template. The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300. This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

Animals↗
PubMed1991

Nuclear magnetic resonance study on the structure and interaction of cyclic AMP receptor protein and its mutants: a deuterium-labeling and photo-CIDNP study.

The identification and assignment of the proton magnetic resonances of some aliphatic and aromatic amino acid residues of cyclic AMP receptor protein (CRP) are reported. The signals of the leucine and valine residues at around 0 ppm were identified on the basis of intermolecular nuclear Overhauser effects, deuterium labeling, and partial proteolytic digestion. On the addition of cAMP, methyl proton signals due to Val-49 and three leucine residues were detected as upfield-shifted signals at around -0.2 ppm. These signals can be used as indicators of the proper binding of cAMP because they are not observed on the addition of cGMP or 2'-deoxy-cAMP. They are also not observed on cAMP binding to mutant CRP*5 (Ser-62-Phe), which can only be activated by a high concentration of cAMP, but they are observed on cAMP binding to other mutant CRP*s (four species), which can be activated by lower concentrations of cAMP. The resonance of some aromatic protons, i.e., C-2H of two tryptophans, C-2H and C-4H of six histidines, and C-2,6H and C-3,5H of six tyrosine residues in CRP, were assigned by means of deuterium labeling and NOE measurements. The 1H NMR spectrum of labeled CRP [Trp(ring-d5), Phe(ring-d5), and Tyr(3,5-d2)] showed good resolution in the aromatic region. The addition of cAMP to this CRP in D2O caused pronounced line broadening of resonances arising from the residues in the cAMP-binding domain, but the resonances of the DNA-binding domain were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Selenium induces changes in the selenocysteine tRNA[Ser]Sec population in mammalian cells.

Two isoacceptors of selenocysteine tRNA[Ser]Sec are present in higher vertebrates which are responsible for donating selenocysteine to protein. One such selenocysteine containing protein, glutathione peroxidase, requires selenium for its translation and transcription. Since tRNA[Ser]Sec is a critical component of the glutathione peroxidase translational machinery, the levels and distributions of its isoacceptors were examined from both human and rat cells grown in chemically defined media with and without selenium. Not only did the level of the selenocysteine tRNA[Ser]Sec population increase approximately 20% in cells grown in the presence of selenium, but the distributions of the two isoacceptors also changed relative to each other.

Animals↗
PubMed1991

DNA and RNA flow cytometric study in multiple myeloma. Clinical correlations.

Flow cytometric studies of cellular DNA and RNA content using the acridine-orange technique were conducted in 81 patients with multiple myeloma (MM). All patients were treated with the M-2 protocol and clinical response was evaluated according to the criteria of the Chronic Leukemia-Myeloma Task Force. Aneuploid DNA stemlines were found in 38.2% of untreated patients with a median DNA index (DNA-I) of 1.15 in marrow aspirates and 1.22 in biopsy specimens. The median percentage of cells with abnormal DNA content was 31.5 (aspirates) and 35 (biopsy specimens) and a positive correlation with the percentage of bone marrow plasma cells was observed. Significantly higher proliferation (S-phase) was found in marrow biopsy specimens as compared with marrow aspirates. Significantly higher RNA content (RNA index [RNA-I]) was observed in aneuploid versus diploid patients in biopsy material. There was no difference in response to the Memorial Hospital M-2 protocol between diploid and aneuploid patients. In patients with DNA-I greater than 1.15 remission duration was shorter as compared with DNA-I less than or equal to 1.15. Furthermore, no difference in cellular RNA content was noted between responders and nonresponders. This study demonstrates no correlation between cellular RNA content and response, as previously described for patients treated with vincristine, Adriamycin, and dexamethasone (VAD), but DNA aneuploidy appears to be an adverse prognostic factor in MM patients treated with the M-2 protocol. It also demonstrates that prognostic models for MM are not universal but depend on the chemotherapeutic regimen used.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Mitoxantrone, etoposide, mitoguazone and vinblastine chemotherapy (MV2) in relapsed and refractory lymphomas.

As part of a program to develop less leukemogenic chemotherapy regimens for the treatment of favorable prognosis Hodgkin's disease, a phase I-II trial of mitoxantrone, etoposide, mitoguazone, and vinblastine was used to treat patients with relapsed and refractory malignant lymphoma and Hodgkin's disease. An overall partial response rate of 41% was observed. Although useful responses were seen, the absence of complete remissions is disappointing.

Adolescent↗