PubMed Health⌕ Search

Biomedical subjects

R L Allen

Publications and source records attributed to R L Allen.

At least 19 recordsLinked to original sources
PubMed1994

Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.

Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.

1-Naphthylisothiocyanate↗
PubMed1994

Identification of T cell receptor recognition residues for a viral peptide presented by HLA B27.

The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383-391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR V alpha 14 gene segment (but not those using TcR V alpha 12) were also sensitive to P1 substitutions, suggesting that the TcR alpha chain of these clones lies over the N terminus of bound peptide, and that the "footprint" of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are "flag" residues with TcR-accessible side chains.

Amino Acid Sequence↗
PubMed1993

Cryptococcal ventricular-peritoneal shunt infection: clinical and epidemiological evaluation of two closely associated cases.

OBJECTIVE: To determine the cause of meningitis associated with Cryptococcus neoformans in two patients with recent ventricular-peritoneal (VP) shunt placement. DESIGN: A retrospective review of materials, records, and concurrent cases of VP shunt procedures. Isolates of C neoformans from each patient were submitted for analysis by colony morphology, biochemical testing, and karyotyping by pulsed-field electrophoresis. SETTING: Two 400-bed community hospitals. PATIENTS: Two immunocompetent patients presented with symptoms of progressive hydrocephalus in August 1991. Each received a VP shunt on the same day by the same surgeon using materials from a common vendor and hospital. RESULTS: Both patients presented within six to eight weeks with symptoms of fever, headache, rash, and cultures of cerebrospinal fluid (CSF) that yielded C neoformans. Each patient recovered after therapy with amphotericin B and flucytosine followed by several months of fluconazole, although one required replacement of the VP shunt for cure. Review of each patient's history and CSF characteristics at the time of shunt placement suggested reactivation of a preexisting infection. Isolates of C neoformans from each patient were submitted for analysis by colony morphology, biochemical testing, and karyotyping by pulsed-field electrophoresis. Each isolate was found to be unique by chromosomal karyotyping. CONCLUSIONS: Our data and previous reports suggest that cryptococcal VP shunt infections appear to be a complication of shunts placed in previously infected persons rather than nosocomial transmission of cryptococcus during placement.

Adult↗
PubMed1993

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development.

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli beta-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.

Base Sequence↗
PubMed1992

Identification of the gene for the developmentally expressed 70 kDa heat-shock protein (P70) of mouse spermatogenic cells.

Mouse spermatogenic cells synthesize a 70-kDa protein (P70) closely related to the major heat-shock protein (hsp70) of mammalian cells (R. L. Allen, D. A. O'Brien, and E. M. Eddy, Mol. Cell. Biol. 8, 828-832, 1988). Expression of P70 is developmentally regulated while hsp70 is induced in response to stress, suggesting that P70 is the product of a unique member of the Hsp70 multigene family transcribed in spermatogenic cells. A strong candidate for this gene was the Hsp70.2 gene (Z. F. Zakeri, D. J. Wolgemuth, and C. R. Hunt, Mol. Cell. Biol. 8, 2925-2932, 1988). A DNA segment from the 5' region of Hsp70.2 hybridized to a 2.7-kb transcript with a temporal pattern of expression in mouse spermatogenic cells similar to the P70 protein. We used a polyclonal antiserum generated against a synthetic peptide predicted from the Hsp70.2 sequence to characterize its protein product and to isolate cDNA clones from a pachytene spermatocyte expression library. The antiserum reacted specifically with meiotic and postmeiotic spermatogenic cells on sections of mouse testis. It recognized the P70 protein on Western blots of two-dimensional gels and did not bind to other heat-shock proteins of spermatogenic or somatic cells. The cDNAs hybridized to a 2.7-kb mRNA that was abundant in unstressed pachytene spermatocytes and round spermatids but was not detected in other cell types. Two cDNAs were sequenced and found to be 99% homologous to the 3' end of the Hsp70.2 gene. These data strongly supported the hypothesis that P70 is the expressed product of the Hsp70.2 gene in mouse spermatogenic cells.

Amino Acid Sequence↗
PubMed1991

Pollen specific cDNA clones from Zea mays.

We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-ripening polygalacturonase and to a pollen-specific polygalacturonase from Oenothera. The other three clones have no significant homologies to any reported sequence.

Amino Acid Sequence↗
PubMed1991

Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.

Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.

Autoradiography↗
PubMed1989

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells.

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.

Adenosine Triphosphate↗
PubMed1988

A novel hsp70-like protein (P70) is present in mouse spermatogenic cells.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.

Animals↗
PubMed1988

Expression of heat shock proteins by isolated mouse spermatogenic cells.

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.

Animals↗
PubMed1987

Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the sulfhydryl-activated toxin of Streptococcus pneumoniae.

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.

Amino Acid Sequence↗
PubMed1986

Phaeohyphomycosis complicating compound skull fracture.

Intracranial infection is a well-recognized complication of compound skull fractures. In most cases various bacteria are identified as the etiologic agents. Fungal infection complicating open head trauma is unusual. We describe a patient who contracted fatal meningoencephalitis due to Drechslera spicifera, a rarely pathogenic soil saprophyte, after open head trauma.

Adult↗
PubMed1986

A comparative study of hepatic and epidermal histidase in the guinea-pig (Cavia porcellus).

Histidine ammonia lyase was purified to homogeneity from guinea-pig liver and epidermis. Both enzymes had similar molecular weights, subunit composition and pH optima. Km values for the two were similar at pH 9.2 but different at pH 7.0. Both enzymes were stimulated by low thiol concentrations and inhibited at higher concentrations, but to different extents. Antibody to the hepatic enzyme showed complete identity against hepatic enzyme but incomplete identity against epidermal enzyme.

Ammonia-Lyases↗
PubMed1986

Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies.

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.

Animals↗