Localization of the neuronal form of nitric oxide synthase to mouse chromosome 5.
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Amino Acid Oxidoreductases↗
Biomedical subjects
C G Lee
Publications and source records attributed to C G Lee.
Metacercarial production of Lymnaea viridis experimentally infected with Fasciola hepatica.
Three groups of Lymnaea viridis snails were infected with one, three or five Fasciola hepatica miracidia, and their metacercarial production was observed. In all groups cercarial shedding commenced in some snails as early as day 27 post-infection. The duration and pattern of cercarial shedding varied from snail to snail in each group. Snails infected with three or five miracidia produced more metacercariae than those infected with a single miracidium. The overall metacercarial production was influenced by the temperature at which the snails were kept during infection.
Animals↗
Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence assay using monoclonal antibody.
Two hybridoma cell lines against Cryptosporidium parvum oocysts (VRI-CN91) were produced. The isotype of these 2 monoclonal antibodies (mAbs) was IgG2b (1E7.2) and and IgM (C6). Enzyme immuno-transfer blotting analysis showed that 1E7.2 reacted specifically to 36 kDa protein and C6 reacted to 67 and 70 kDa proteins. C. parvum was bound specifically to the surface region of oocysts by these mAbs. No cross-reactivity was observed with tachyzoites of Toxoplasma gondii and oocysts of Eimeria zuernii, E. bovis and E. canadensis of bovine origin. The indirect immunofluorescence assay (IIF) using mAb C6 was successful with counterstain. With the IIF using mAb C6, oocysts appeared as 3 to 5 microns spherical objects fluorescing bright apple green against a reddish dark background. The IIF using mAb C6 was agreed in specificity and sensitivity with those of a commercial diagnostic kit. These results demonstrated that the produced mAbs were specific to C. parvum and that the mAb C6 could be used for diagnosis of cryptosporidiosis.
Animals↗
[Changes of arterial oxygen tension in supine position during one-lung anesthesia].
BACKGROUND: One-lung ventilation during anesthesia (one-lung anesthesia) in patients under lateral decubitus position to help performing intra-thoracic surgical procedures was well known to have larger alveolar-to-arterial oxygen tension difference and lower arterial oxygen tension (PaO2) as compared to two-lung ventilation. In the present study, we investigate the changes of arterial oxygen tension in the supine position during one-lung anesthesia. METHODS: Forty-two patients of palmar hyperhidrosis, ASA class I-II, scheduled to receive bilateral transthoracic endoscopic sympathectomy were studied. After anesthetic induction (fentanyl, thiopental, and succinylcholine), a 35 (for female) or 37 (for male) French left-sided Robertshaw double-lumen endobronchial tube was intubated. Anesthesia was maintained with isoflurane 1.0-1.5% and 50% O2-N2O. They were changed to isoflurane 1.5-2.0% and 100% O2 during one-lung ventilation. Once the surgical operation is completed, they were changed to 100% O2 and two-lung ventilation. Arterial blood gases were measured at 4 phases: 5 min after endobronchial intubation (two-lung ventilation), 5 min after left one-lung ventilation, 5 min after right one-lung ventilation, and 5 min after accomplishing operation (two-lung ventilation). RESULTS: The results showed PaO2 were significantly lower in left and right one-lung ventilation with 100% O2 as compared with that obtained from two-lung ventilation with 50% O2 5 min after endobronchial intubation (p < 0.05). Furthermore, right one-lung ventilation had a lower PaO2 than left one-lung ventilation (p < 0.05). CONCLUSIONS: We conclude that arterial oxygen tension can be safely maintained during one-lung anesthesia with pure oxygen in healthy patients lying in a supine position.
Adolescent↗
Molecular cloning and characterization of a murine LPS-inducible cDNA.
Murine macrophages respond to endotoxins by inducing a vast array of genes that play a major role in the host's response to infection and tumor growth. We have isolated and characterized a 1.8-kb cDNA, designated IRG2, from a cDNA library prepared from RNA isolated from the murine cell line, RAW 264.7, after bacterial LPS stimulation. The cDNA encodes a protein of 47 kDa that is the murine homologue of a small family of proteins described from IFN-induced human cells. The IRG2 message does not appear until 3 h after LPS exposure and its induction is dependent on new protein synthesis. IRG2 induction by LPS is slightly inhibited by the anti-inflammatory steroid, dexamethasone. Increasing cytosolic cAMP with either forskolin, dibutyryl cAMP, or 8-(4-chlorophenylthio)-cAMP caused marked inhibition of the LPS induction of IRG2. In contrast, activation of PKC with phorbol ester potentiated the LPS response. Removing extracellular Ca2+ with EGTA inhibited IRG2 induction; increasing intracellular calcium with the calcium ionophore A23187 led to enhanced levels of the IRG2 transcript. These data suggest that the induction of IRG2 occurs via a PKC pathway.
Amino Acid Sequence↗
Localization of a novel, LPS-inducible member of the thymidylate kinase family to mouse chromosome 12.
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Animals↗
Rapid growth of Lymnaea viridis, the intermediate host of Fasciola hepatica, under laboratory conditions.
Reported in this communication is a unusually rapid growth of Lymnaea viridis, the intermediate host of Fasciola hepatica, maintained in the laboratory for experimental purposes. When the snails were fed on blue-green algae at about 20 degrees C, they attained a shell length of 12 mm in 37 days from the date of hatching. The snails began laying eggs from 18 to 24 days following hatching.
Animals↗
[Isolation of Cryptosporidium parvum oocysts from fecal samples--the combination of ether extraction and discontinuous sucrose gradients].
A calf and 50 mice were infected with Cryptosporidium parvum, and their fecal materials were collected and treated with ether extraction (EE), followed by discontinuous sucrose gradients (DSG) method. EE method was to remove some of fat or lipid from feces. Sediments were washed by centrifugation (1,500 x g for 10 min., 3 times) in phosphate-buffered saline and then these washed sediments were sieved sequentially through stainless steel screens with a final mesh of 250 (61 microns porosity) to remove other debris. After sieving, the materials were suspended in 2.5% potassium dichromate solution. Oocysts were counted by using a hemocytometer and the recovery rate of pure oocysts was calculated on the basis of the count. Following centrifugation (1,500 x g for 30 min.) by DSG method, most oocysts were recovered at the interface between a gravity of 1.103 and 1.064. The recovery rates of pure oocysts from the fecal suspension of the calf (3.8 x 10(7)/ml) and the mouse (3.2 x 10(6)/ml) treated with EE method were 81.6% and 51.6%, respectively. It is suggested that the recovery rate was dependent on the number of oocysts in each suspension treated with EE method. To get the 50% recovery rate, there must be more than 2 x 10(6) oocysts per ml of the fecal suspension treated with EE method. By the combination of the two methods it was possible to isolate C. parvum oocysts from normal feces of the calf and mouse as well as from diarrheic feces.
Animals↗
Active DNA topoisomerase II with minimum molecular mass from regenerating rat liver.
We have purified the type II DNA topoisomerase from regenerating rat liver. The purified topoisomerase II migrated as two bands with molecular masses of 70 kDa and 55 kDa on SDS-PAGE. Immunoblotting analysis using antiserum against rat topoisomerase II gene product expressed in Escherichia coli suggested that the two bands on SDS-gel are proteolytic products of the intact 173 kDa form. However, these products retained the enzyme activities such as catenation and relaxation of supercoiled circular duplex monomer DNA and unknotting of knotted phage P4 DNA. These results suggest that DNA topoisomerase II consists of different functional domains and that the whole enzyme is not required for its activity. The activities of the purified enzyme were completely inhibited by 1 mM novobiocin, a bacterial gyrase inhibitor. However, no inhibitory effect was observed when another gyrase inhibitor, nalidixic acid was used.
Animals↗
Allosteric interaction of a herpes simplex viral thymidine kinase with host DNA polymerase alpha in mouse LP1-1 cells.
A DNA polymerase alpha-associated multienzyme complex isolated from mouse LP1-1 cells transfected with the thymidine kinase gene of herpes simplex virus type I (1) showed activities of DNA polymerase alpha, topoisomerase II, and thymidine kinase (TK) in the complex. TK antiserum recognized a 43 kDa polypeptide only in the fraction of the multienzyme complex prepared from the LP1-1 cells but not that from L-M(TK-) cells. In permeabilized cells, hydroxyurea did not show any inhibitory effect on either DNA polymerase or TK, whereas aphidicolin, novobiocin, and TK antiserum inhibited both enzymes. These results provide evidence for the functional association and an allosteric interaction between the viral TK and host DNA polymerase alpha.
Allosteric Regulation↗
Human RNA helicase A is homologous to the maleless protein of Drosophila.
RNA helicase A is an abundant nuclear enzyme of HeLa cells that unwinds double-stranded RNA in a 3' to 5'direction (Lee, C. G., and Hurwitz, J. (1992) J. Biol. Chem. 267, 4398-4407). A complementary DNA (cDNA) clone expressing RNA helicase A was isolated by screening a human cDNA library with polyclonal antibodies produced against the purified protein. The deduced amino acid sequence from this clone showed that RNA helicase A is a member of the DEAH family of proteins thought to be helicases. Sequence comparison among all known proteins of the DEAH family revealed that the highest homology was between RNA helicase A and the maleless protein (MLE) of Drosophila. There was 49% identity and 85% similarity throughout the overall primary sequences of both proteins, suggesting that RNA helicase A is the human counterpart of Drosophila MLE. Polyclonal antibodies against Drosophila MLE recognized RNA helicase A in crude nuclear extracts of HeLa cells as well as the purified protein. A recombinant RNA helicase A containing 6 histidine residues at the NH2 terminus was expressed in Sf9 cells using a baculovirus vector. The protein isolated from insect cells and the enzyme purified from HeLa cells exhibited identical RNA helicase and RNA-dependent ATPase activities.
Amino Acid Sequence↗
RNA annealing activity is intrinsically associated with U2AF.
U2AF is a protein that is essential for the formation of the prespliceosome complex during pre-mRNA splicing. It contains two subunits, 65 and 35 kDa, although only the 65-kDa subunit has been shown to be essential for its splicing activity. Here, we show that the 65-kDa subunit mediates the annealing of complementary single-stranded RNAs or single-stranded DNAs. This activity was shown to reverse the action of RNA helicase A, an enzyme that catalyzes the displacement of duplex RNAs. The NH2-terminal region of the 65-kDa subunit of U2AF, containing arginine-serine (RS) dipeptides and basic amino acid sequences, was shown to be essential for the annealing of complementary sequences, RNA binding, and the inhibition of RNA helicase A activity. Thus, through the combined action of U2AF and RNA helicases, duplex RNA regions can be reversibly formed and displaced. Such reactions appear to be critical for pre-mRNA splicing, translation, and transcription.
Base Sequence↗
Evidence that 68-kilodalton and 54-51-kilodalton polypeptides are components of the human sperm fibrous sheath.
This study characterizes a common antigen recognized by two monoclonal antibodies (mAbs) that immunoreact with the principal piece of the human sperm flagellum. By means of immunofluorescence microscopy, mAbs S69 (IgM) and S70 (IgG1) (WHO Task-force nomenclature) were observed to bind to the principal piece of methanol- or detergent-permeabilized human spermatozoa, but did not react with live swimming spermatozoa as assessed by immunofluorescence microscopy. Faint immunofluorescence was also seen on the connecting piece in approximately 40% of the spermatozoa. Immunoreactivity in both regions was resistant to sequential extraction with Triton X-100, sodium thiocyanate, and urea. Pre-embedding electron microscopic immunogold labeling of ejaculated spermatozoa with mAb S69 showed gold particles located on the fibrous sheath. Immunoreactive peptides of 68, 53, and 45 kDa were recognized by both S69 and S70 mAbs on immunoblots of nonreduced human sperm extracts, while a 68-kDa band and a strongly immunoreactive triplet from 54 to 51 kDa were recognized in reduced sperm extracts. Human fibrous sheaths were isolated by differential solubilization and centrifugation and characterized by transmission electron microscopy. The 68-kDa and 54-51-kDa bands were enriched and found to be major polypeptides in the isolated fibrous sheath fraction. These results suggest that the S69/S70 antigen, which we term SP (sperm protein) (68 kDa, 54-51 kDa), is a component of the human fibrous sheath.
Animals↗
Banding patterns of Fasciola hepatica and Fasciola gigantica (Trematoda) by isoelectric focusing.
Liver flukes Fasciola hepatica and Fasciola gigantica are polymorphic and vary morphologically depending upon the host being parasitized. It is known also that mixed infection occurs where both species are present. A technique involving protein separation was used to distinguish the 2 species. Isoelectric focusing of soluble proteins was performed on polyacrylamide gels using whole-body proteins from adult flukes. Although many bands appeared common to both species and some were shared with host tissues, the banding patterns could be used to distinguish 1 species from the other. Soluble protein isoelectric focusing is simple, reproducible, and has very good resolution. It seems well suited to the differentiation of the 2 fluke species.
Animals↗
Selective 5' modification of T7 RNA polymerase transcripts.
We have developed two methods for selective 5' modification of RNAs generated by enzymatic synthesis using T7 RNA polymerase. The first method involves a two-step procedure. Transcription reactions are performed under standard conditions except that GTP is replaced by GTP gamma S. Since the polymerase initiates transcription with GTP, every transcript contains a 5' gamma-thiophosphate group which is modified with the thiol-specific reagent of choice (e.g., iodoacetyl dansyl derivative) in the second step. In an alternative method, transcription and modification reactions are carried out in a single step, using a mixture of dansylated GTP and GTP. Under the appropriate conditions, dansylated GTP effectively competes with GTP in the initiation reaction but does not substantially inhibit the elongation reaction. Yields of fluorescent 64-mer RNA ranging from 30 to 70% of the total transcription product have been obtained using these methods in combination with HPLC purification. This approach is amenable to large scale synthesis reactions and can be used to produce a wide variety of 5'-modified RNAs of virtually any size for structural or functional studies.
Base Sequence↗
A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction.
We have purified an RNA helicase to near homogeneity from nuclear extracts of HeLa cells. The enzyme migrated as a 130-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and exhibited a sedimentation coefficient of 6.4 on glycerol gradient centrifugation. The enzyme translocated in a 3' to 5' direction and acted catalytically, displacing at least a 4-fold molar excess of duplex RNA compared with the enzyme added. All eight common nucleoside triphosphates supported RNA helicase activity at relatively low concentrations (Km in values in the 15-20 microM level). In the presence of RNA and some single-stranded DNAs, the RNA helicase hydrolyzed all nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate. The enzyme displaced deoxyribooligonucleotides provided they were hydrogen-bonded to RNA possessing 3' single-stranded regions, but it did not displace ribooligonucleotides hydrogen-bonded to DNA containing 3' single-stranded regions. The enzyme, in the absence of ATP, binds to both single-stranded RNA and DNA, but the amount of complex formed with RNA was 20-fold greater than the complex formed with DNA. In both cases, the complex formed in the absence of ATP was rapidly reversed by the addition of ATP and not by adenyl-5'-yl (beta,gamma-methylene)-diphosphate. We propose that the enzyme can bind to both single-stranded RNA and DNA and hydrolyze ATP, but by virtue of its greater stability on RNA, the enzyme can only translocate on RNA possessing 3' single-stranded regions.
Adenosine Triphosphate↗
Host influence on the banding profiles of whole-body protein and excretory-secretory product of Fasciola hepatica (Trematoda) by isoelectric focusing.
Various protein separation techniques have been used as alternative means of differentiating species and strains of parasites. Of these techniques, isoelectric focusing (IEF) has proved to be specific and reproducible with cestodes, protozoa and trematodes. As with the traditional morphological approach, there is, however, the potential for host influence. For a particular separation technique to be useful, it is important that banding profiles differentiating parasites be consistent, regardless of host species. To test for host influence, Fasciola hepatica were maintained in four definitive hosts. Soluble proteins of flukes from different hosts and their respective excretory-secretory product (ESP) were then subjected to IEF together with respective host tissues, and the resulting banding patterns were compared. Protein banding profiles of fluke body protein from llama, rat and mouse were very similar, while those of flukes from calves were distinct. Banding patterns of fluke ESP from different hosts were dissimilar. Overall banding patterns of body protein and ESP of flukes from different hosts were distinct from those of respective host tissues. The unique banding patterns of the body protein of flukes from calves indicate that there was host influence in their protein banding patterns, and the uniqueness of the calf fluke is discussed relative to host's resistance to the fluke.
Animals↗
Fasciola hepatica: comparison of flukes from Korea and the United States by isoelectric focusing banding patterns of whole-body protein.
Comparisons were made between the flukes from Chonnam, Korea and Oregon, USA by isoelectric focusing (IEF) of whole-body protein. Adult Fasciola hepatica were recovered from bile ducts of Korean native cattle. Whole-body protein of the flukes was subjected to IEF, and the banding patterns of the fluke protein were compared with those of North American F. hepatica recovered from experimentally infected calves. The overall banding pattern of F. hepatica from Korea was essentially identical to that of F. hepatica from the United States. These results provide further support for the usefulness of this technique in differentiating Fasciola species in other geographical areas.
Animals↗