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Biomedical subjects

R H Miller

Publications and source records attributed to R H Miller.

At least 235 records · Page 13Linked to original sources
PubMed1989

Complete nucleotide sequence of a molecular clone of woodchuck hepatitis virus that is infectious in the natural host.

Woodchuck hepatitis virus (WHV) DNA was cloned from viral particles obtained from the serum of a woodchuck with a naturally acquired infection. The complete nucleotide sequence of the virus genome was determined and found to be 3323 base pairs long. Transfection experiments demonstrated that the recombinant WHV DNA was infectious in each of 18 woodchucks tested and established a chronic carrier state in 1 of 13 neonates and 3 of 5 adult animals. WHV DNA from serum particles from the chronically infected neonate was cloned and the nucleotide sequence of three independent recombinants was compared directly with that of the input recombinant DNA. The consensus sequence of the three progeny genomes was identical to that of the parental DNA sequence. Therefore, transfection of woodchuck livers with recombinant WHV DNA induces active virus replication and gene expression and yields progeny genomes that are faithful copies of the input virus genome.

Animals↗
PubMed1989

One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

Cations, Divalent↗
PubMed1989

Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique.

We have developed a rapid procedure for the detection of serum hepatitis B virus (HBV) DNA using the polymerase chain reaction (PCR) technique. HBV DNA is released from virions by incubating serum with 0.1 M NaOH for 60 min at 37 degrees C. The mixture is brought to neutral pH with HCl, and the HBV DNA sequences are detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with two successive sets of primer pairs. The detection limit of this method (i.e., 10(-5) pg of HBV DNA) is equivalent to that previously determined by one round of PCR amplification and Southern blot hybridization analysis. The advantages are that the assay can be completed in 1 day, is very sensitive, and does not require the use of radiolabeled reagents.

Blotting, Southern↗
PubMed1989

Laryngeal trauma. Diagnosis and management.

Acute laryngeal trauma caused by either blunt or penetrating injuries is a difficult airway management problem. Patients may be aphonic or intubated due to the injury, and important historical facts may not be available. Because of this, the recognition and initial proper management of this injury is essential to a successful outcome. Flexible endoscopy provides an easy and accurate examination of the injured larynx without risk of injury to the patient. Assessment of vascular and esophageal injuries is also the key in management of this patient group. The use of CT imaging helps delineate injuries that are not well defined by physical examination. Immediate operative intervention with repair of mucosal injuries and stabilization of cartilaginous framework usually results in good airway and voice quality.

Humans↗
PubMed1988

Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand.

The genome of the human immunodeficiency virus (HIV) is known to contain eight open reading frames (ORFs) on the minus strand of the double-stranded DNA replicative intermediate. Data presented here indicate that the DNA plus strand of HIV contains a previously unidentified ORF in a region complementary to the envelope gene sequence. This ORF could encode a protein of approximately 190 amino acid residues with a relative molecular mass of 20 kilodaltons if translation began from the first initiation codon. The predicted protein is highly hydrophobic and thus could be membrane associated. It is possible, therefore, that the HIV genome encodes a protein on antisense messenger RNA.

Amino Acid Sequence↗
PubMed1988

Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. Immunochemical analysis of the enzyme complex.

Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1988

Anaplastic thyroid carcinoma. Association with differentiated thyroid cancer.

Fourteen cases of anaplastic thyroid carcinoma (ATC) treated at Baylor College of Medicine, Houston, during a 19-year period were reviewed. There was a 1.3:1 female preponderance, and the median patient age was 62 years. Ten (71%) of the anaplastic tumors either followed or occurred simultaneously with a well-differentiated thyroid carcinoma. This finding adds support to the theory that ATC usually results from the transformation of well-differentiated thyroid carcinoma. A combination of surgery, radiation therapy, and chemotherapy was utilized for most patients. In this series of patients, the median duration of survival was four months following diagnosis, with only three patients (21%) alive at one year. Despite aggressive therapy, the prognosis for ATC remains poor.

Adult↗
PubMed1988

Evidence that migratory oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells are kept out of the rat retina by a barrier at the eye-end of the optic nerve.

There is evidence that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells migrate along the developing rat optic nerve from the chiasm toward the eye before differentiating into oligodendrocytes that myelinate the retinal ganglion cell axons in the nerve. Why, then, do these progenitor cells not migrate into the eye, differentiate into oligodendrocytes and myelinate the nerve fibre layer of the retina? Myelination would opacify the neural retina and thereby severely impair vision. Here we provide evidence that there is a barrier at the eye-end of the rat optic nerve that prevents the migration of O-2A progenitor cells into the retina. Our findings in the rat support a previous hypothesis that such a barrier keeps myelin-forming glial cells out of the human retina.

Animals↗
PubMed1988

The epidemiology of the postpericardiotomy syndrome: a common complication of cardiac surgery.

PPS is a major cause of morbidity after cardiac surgery and may cause bypass graft closure and fatal cardiac tamponade. Little is known about its incidence and cause. To better define this syndrome characterized by postoperative fever, pericardial friction rub, and pericardial pain, we used two out of three of the preceding criteria to diagnose PPS. In a prospective epidemiologic study we followed 944 consecutive patients undergoing open-heart surgery between November 1984 and November 1985. The overall incidence was 17.8%. The incidence was increased in younger patients, in those with a history of prednisone use in the past, in patients with a past history of pericarditis, those with aortic valve replacement, and in patients who received enflurane or halothane anesthesia. PPS is a common syndrome. Knowledge of risk factors associated with PPS may allow its prevention and identification of patients who warrant early and aggressive treatment.

Age Factors↗
PubMed1988

Sequence comparison of woodchuck hepatitis virus replicative forms shows conservation of the genome.

The complete nucleotide sequence of virion DNA from two isolates of woodchuck hepatitis virus (WHV 7 and WHV 59) was determined along with the sequence of supercoiled DNA from one of those isolates (WHV 7). The sequences of the two WHV isolates were compared with the previously published sequences of two other isolates (WHV 1 and WHV 8). The range of nucleotide sequence variation of the four isolates (1 to 3.5%) was similar to those of HBVs of the same subtype (1.5 to 2%), but less than those of HBVs of different subtypes (8 to 10%). When amino acid sequences from the four WHV isolates were aligned, a consensus sequence could not be obtained at 13 sites. At 11 of 13 sites (7 in polymerase, 2 in presurface, 1 in surface, and 1 in the X region) the viruses could be grouped into pairs, so that WHV 1 and WHV 59 shared one amino acid and/or WHV 7 and WHV 8 shared a different amino acid. WHV 7 was passaged twice in woodchucks and supercoiled DNA was isolated. The nucleotide sequence of WHV 7 supercoiled DNA (derived from liver) was compared to that of WHV 7 virion DNA (derived from serum). Sequence comparison of virion and free supercoiled DNA from WHV 7 showed no nucleotide changes, except for a single nucleotide deletion thought to represent an error during molecular cloning or a defective viral genome. Thus, the nucleotide sequence of two different replicative forms of WHV DNA, separated by two in vivo passages, were virtually identical.

Amino Acid Sequence↗
PubMed1988

Close evolutionary relatedness of the hepatitis B virus and murine leukemia virus polymerase gene sequences.

Previous work indicates that hepatitis B virus (HBV) and retroviruses utilize a unique mechanism for genome replication by reverse transcription of RNA and share homology in biologically important nucleotide and protein sequences. The data presented here extend previous findings of sequence homology among the genomes of the members of these virus families. HBV was found to possess sequences homologous to the retrovirus protease and reverse transcriptase gene sequences. Homology was not found to the retrovirus integrase sequence consistent with the observation that hepadnaviruses do not integrate into cellular DNA as a necessary step in their replication cycle. Overall, the homology of the hepadnavirus polymerase gene was strongest with that of the murine leukemia viruses (MLVs). Also, the hepadnavirus polymerase shares organizational similarities to the MLV polymerase sequence. Analysis suggests that the ancestor of both hepadnaviruses and retroviruses possessed an overlapping long open reading frame in the polymerase gene sequence. In addition, low stringency blot hybridization using hepadnavirus DNA probes indicates that HBV is more closely related to MLV sequences than the sequences of MLV-related viruses and endogenous retrovirus-like genetic elements. Taken together, the data indicate that the polymerase gene sequence of the hepadnavirus and MLV genomes are organized in a similar fashion which suggests that these viruses evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1988

Choanal atresia and associated anomalies: the CHARGE association.

Over the past few years there has been increasing awareness of the association of choanal atresia with other congenital defects. Thirty-one cases of choanal atresia were reviewed and other congenital anomalies were documented in 19 patients. These other anomalies were identified in 75% of the 20 patients with bilateral choanal atresia but in only 36% of the 11 patients with unilateral choanal atresia. Associated defects largely occurred in a predictable pattern described by the CHARGE mnemonic: C-coloboma, H-heart disease, A-atresia choanae, R-retarded growth, G-genital hypoplasia, E-ear defects. The implications of the CHARGE association in the management of patients with choanal atresia are discussed.

Abnormalities, Multiple↗
PubMed1988

X gene-related sequences in the core gene of duck and heron hepatitis B viruses.

The genomes of all known mammalian hepadnaviruses contain an open reading frame (ORF), designated X, located just upstream from the gene encoding the major viral nucleocapsid polypeptide. This gene is believed to have one or more roles central to the life cycle of these viruses. Consequently, it is surprising that avian hepadnaviruses appear to lack this ORF. However, the observation that the size and position of the core gene in the duck hepatitis B virus (DHBV) genome and the heron hepatitis B virus (HHBV) genome is comparable to the combined X and core genes of the mammalian hepadnaviruses suggests that X function(s) may be performed by the major nucleocapsid polypeptide of DHBV and HHBV. Computer-assisted analyses were carried out to test the hypothesis that the primary and secondary structural characteristics of the X gene product are also present in the major core gene product of the duck (DHBcAg) and heron (HHBcAg) viruses. Primary sequence comparison of the major core-associated polypeptides encoded by the avian and mammalian hepadnaviruses demonstrates considerable homology at both the amino- and carboxyl-terminal regions of these components. However, the middle portion of the DHBcAg and HHBcAg polypeptide, spanning about half the molecule, is unique. Comparison of this region with the carboxyl-terminal half of the X gene sequences from mammalian hepadnaviruses demonstrates similarities in both primary sequence and secondary structural characteristics. These results suggest that X-like gene product sequences are present in the core gene products of DHBV and HHBV. In addition, a sequence of about two dozen residues at the amino terminus of the mammalian X gene product, overlapping the polymerase gene product, is found in the corresponding position in DHBV. This is consistent with the conclusion that the relationship between the DHBV and HHBV core genes compared to the X and core genes of the mammalian hepadnaviruses may be explained by one or more translocations in the this region of the viral genome. The previous finding of X antigen determinants associated with one or more core-related polypeptides in the mammalian hepadnaviruses, combined with the results of this study, suggests that X gene product function is conserved among these viruses.

Amino Acid Sequence↗
PubMed1988

X-region-specific transcript in mammalian hepatitis B virus-infected liver.

In vitro gene expression systems for hepatitis B virus have demonstrated that the virus genome is capable of producing an X-region-specific transcript of approximately 0.7 kilobases (kb). However, this transcript has not been detected in virus-infected cells. We now report the presence of a heterogeneous X-region-specific transcript of approximately 0.65 kb that is found primarily in the nucleus of liver cells infected with the woodchuck hepatitis virus. Interestingly, the majority of the transcripts are not polyadenylated. The transcript, which represents less than 1% of total virus-specific RNA, is found in animals with both acute and chronic woodchuck hepatitis virus infections. While it is probable that the 0.65-kb transcript is involved in the expression of the X gene protein, it may also direct the translation of a protein encoded by a newly identified open reading frame, ORF5, that is present in all hepadnavirus genomes analyzed.

Animals↗