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Biomedical subjects

M E John

Publications and source records attributed to M E John.

At least 37 records · Page 2Linked to original sources
PubMed1992

Effect of a superficial and a deep scleral pocket incision on the incidence of hyphema.

Deep tunnel scleral pocket incisions were developed to control surgically induced astigmatism following cataract and intraocular lens implantation surgery. A more superficial, shallow scleral pocket incision was developed to reduce the rate of postoperative hyphema. To test its effectiveness, a randomized prospective clinical study was performed. One hundred twenty-nine eyes of 129 patients were randomized: 66 to receive a deep tunnel pocket and 63 to receive the superficial pocket. In both groups the incision was made 3 mm posterior to the limbus. For the deep pocket cases, a blade setting of 0.27 mm was used and the wound was dissected forward toward the entry site, creating a long, deep, narrow tunnel. For the superficial wound cases, a 0.17 mm blade setting was used and the entire wound was dissected to the edge of the anatomic limbus, creating a thin scleral flap. The left side was then fanned out toward the left with the entry site slightly into clear cornea. At one day after surgery, 22 cases (34%) in the deep pocket group and four cases (6%) in the superficial pocket group had hyphemas. The difference in hyphema rates was statistically significant (P less than .001).

Adult↗
PubMed1990

Sequence analysis of arcelin 2, a lectin-like plant protein.

The nucleotide sequence of the cDNA clone encoding arcelin 2 (Arc2), one member of a family of closely related lectin-like plant toxins from wild bean accession, is presented. The sequence contains a 265-amino acid (aa) open reading frame and is 99.3% homologous to Arc1, another of the four electrophoretic variants with proven antibiosis characters. These two proteins differ by four aa residues. Based on cross hybridizations of RNAs, it is assumed that Arc4 is more divergent than Arc1 and Arc2. Furthermore, it is likely that at least three of the variants are polypeptides of similar size and the observed molecular weight differences between them are due to differences in the number of glycosylation sites.

Amino Acid Sequence↗
PubMed1989

Effect of ACTH on the stability of mRNAs encoding bovine adrenocortical P-450scc, P-45011 beta, P-45017 alpha, P-450C21 and adrenodoxin.

ACTH treatment of bovine adrenocortical cells in primary culture causes increased accumulation of mRNAs encoding cytochromes P-450SCC, P-450(11)beta, P-450(17) alpha, P-450C21 and adrenodoxin as well as increased transcriptional activity of their respective genes. In this study we have shown that ACTH does not greatly affect the half-life of mRNAs encoding P-450(11)beta, P-450(17)alpha, P-450C21 and adrenodoxin. However, in the case of P-450SCC mRNA, ACTH causes a five-fold increase in the half-life leading to a significant stabilization of P-450SCC mRNA. Thus it appears that the levels of mRNAs encoding P-450(11)beta, P-450(17)alpha, P-450C21 and adrenodoxin are regulated by ACTH primarily at the transcriptional level, while that for P-450SCC is regulated at both the transcriptional and post-transcriptional levels.

Adrenal Cortex↗
PubMed1989

Structural characterization of the bovine CYP17 (17 alpha-hydroxylase) gene.

The complete exonic and partial intronic sequence of the bovine CYP17 (P45017 alpha) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when approximately 500 bp of the 5'-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated.

Adrenal Cortex↗
PubMed1989

Advanced versus immature cataracts--a preliminary report.

We did a prospective clinical study to determine whether removing cataracts which had reached an "advanced" stage were: (1) more difficult to remove, or (2) resulted in decreased postoperative visual acuity compared with immature cataracts. Our data showed that 48.5% of the advanced cases were more difficult to remove than immature cataract cases (17%). The presence of "peripheral lacy cortex" occurred 37 times more frequently in the advanced group than in the immature group. These two parameters might explain the difference in postoperative visual acuity where only 79% of the advanced and 92% of the immature cataracts had 20/40 visual acuity or better (P less than 0.01). Our results suggest that patients should be informed of the increased risks in waiting to have surgery done.

Adult↗
PubMed1987

Regulation of the biosynthesis of steroidogenic enzymes.

Recombinant DNA technology can permit study of the regulation of steroid hydroxylase gene expression at three levels. The first of these is cAMP-regulated gene expression. In the adrenal, ACTH, via cAMP, increases the expression of the genes for all of the cytochrome P-450 species involved in the steroid biosynthetic pathway, as well as the iron-sulfur protein, adrenodoxin. This action of cAMP is inhibited by cycloheximide, suggestive of the involvement of a regulatory protein factor in mediating this action of cAMP. The second level is tissue-specific regulation of steroid hydroxylase gene expression. An example of this which we have studied is the expression of cholesterol side-chain cleavage cytochrome P-450 (P-450sec) and 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) in the bovine ovary. P-450sec is expressed at high levels in the corpus luteum but at low levels in follicles, whereas P-450(17)alpha is expressed in follicles, but is undetectable in the corpus luteum. The third level is fetal imprinting. A number of the cytochrome P-450 species involving in the steroidogenic pathway are expressed in the fetal adrenal at a time when exposure of the gland to ACTH is very low, suggestive that factor(s) other than pituitary ACTH mediate this expression in fetal life.

Adrenal Glands↗
PubMed1987

Ontogeny of adrenal steroid hydroxylases: evidence for cAMP-independent gene expression.

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450scc, P-450(11) beta, P-450(17) alpha, P-450C21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450scc, P450(17) alpha and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85-145 days) showed constant levels of P-450scc and P-450(11) beta, but increasing P-450(17) alpha content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.

Adenylyl Cyclases↗
PubMed1986

Bovine steroid 21-hydroxylase: regulation of biosynthesis.

A recombinant cDNA clone, PBC21-1, specific for bovine steroid 21-hydroxylase cytochrome P-450 (P-450C21) was identified in a bovine adrenocortical cDNA library, and this identity was confirmed by nucleotide sequencing which revealed significant amino acid homology (77%) with human P-450C21 cDNA. The pBC21-1 insert is 1.7 kilobases in length and includes a 1128 base pair region that encodes the C-terminal 376 amino acids of bovine P-450C21 as well as 535 base pairs of 3'-untranslated sequence. A novel feature of this insert is a 20 base pair intervening sequence near the 5' end, apparently the result of an aberrant splicing event. Northern blot analysis reveals that bovine P-450C21 is encoded by two transcripts, 2.3 and 2.0 kilobases in length which are detected in adrenal cortical RNA. Bovine liver, heart, kidney, and corpus luteum do not contain detectable P-450C21 transcripts. Regulation of P-450C21 gene expression by adrenocorticotropin was investigated with pBC21-1 and bovine adrenocortical cells in primary, monolayer culture. Treatment with ACTH or analogues of cAMP increases the steady-state levels of P-450C21 RNA in such cell cultures. In vitro transcription run-on assays suggest that this increase is, at least in part, due to the enhanced transcriptional activity of the P-450C21 gene.

Adrenal Cortex↗
PubMed1986

Bovine adrenocortical cytochrome P-450(17 alpha). Regulation of gene expression by ACTH and elucidation of primary sequence.

In the steroidogenic pathway of the adrenal cortex, 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) is a major regulatory enzyme. Previous studies have shown that stimulation of 17 alpha-hydroxylase activity occurs upon treatment of bovine adrenocortical cells in culture with adrenocorticotropic hormone (ACTH), via cAMP, due to an increase in the enzyme concentration. Alterations in the levels of this enzyme result in pronounced changes in the pattern of steroids produced, with the potent glucocorticoid cortisol, as well as the sex steroids, being products of 17 alpha-hydroxylated steroid precursors. In the present study, the identification and sequencing of two cloned DNA molecules, pB17 alpha-1 and pcD17 alpha-2, which are complementary to mRNA sequences encoding bovine adrenal cortex P-450(17 alpha), are reported. Clone pcD17 alpha-2 contains an open reading frame coding for the complete amino acid sequence of P-450(17 alpha) which consists of 509 amino acids and has a molecular weight of 57,251. By comparison to other forms of cytochrome P-450, we conclude that P-450(17 alpha) is a member of a previously unidentified family within the P-450 multigene superfamily. Single bovine and human mRNA species of approximately equal to 1850 bases in length hybridize to these clones. Regulation of the concentration of this RNA has been studied using bovine adrenocortical cells in culture. Treatment with ACTH or dibutyryl cAMP causes an increase in the content of P-450(17 alpha) RNA in as little as 2 h, and its concentration increases greater than 20-fold by 8 h. In contrast, other steroidogenic enzymes that have been studied respond more slowly to ACTH. Actinomycin D and cycloheximide block induction of P-450(17 alpha) RNA, indicating that regulation of 17 alpha-hydroxylase activity is controlled at the level of transcription and requires ongoing protein synthesis.

Adrenal Cortex↗
PubMed1986

Regulation of cholesterol side-chain cleavage cytochrome P-450 gene expression in adrenal cells in monolayer culture.

Utilizing a cDNA probe specific for bovine cytochrome P-450scc and primary monolayer cultures of bovine adrenocortical cells and human fetal adrenal cells, it has been shown that the chronic action of ACTH on the adrenal cortex includes regulation of P-450scc gene expression at the transcriptional level. The bovine P-450scc cDNA hybridizes strongly to human, pig and rat RNA. Advantage was taken of the cross-reactivity of the bovine P-450scc cDNA with human P-450scc RNA to examine the regulation of P-450scc gene expression by ACTH in human fetal adrenal cells. This process is mediated by cyclic AMP and is inhibited by cycloheximide, in a fashion similar to bovine adrenocortical cells, suggestive that a protein factor(s) activates the response in both species. Hence, the actions of ACTH to regulate P-450scc gene expression in bovine adult adrenocortical cells and human fetal adrenal cells appear to proceed by similar mechanisms.

Adrenal Glands↗
PubMed1986

Transcriptional regulation of steroid hydroxylase genes by corticotropin.

Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues.

Adrenal Cortex↗
PubMed1986

Control of gene expression of adrenal steroid hydroxylases and related enzymes.

Utilization of cDNA probes specific for various components of the bovine adrenocortical steroidogenic pathway have led to the conclusion that there are three levels of regulation of steroid hydroxylase gene expression. In each case it is postulated that specific classes of proteins bind to regulatory regions of these genes and modulate their transcription. Throughout adult life, cAMP-dependent regulation via SHIP protein(s) is the predominant mechanism by which optimal steroidogenic capacity is maintained. A second type of regulation is tissue-specific. One subclass of tissue-specific expression is the "all-or-none" type whereby steroid 21-hydroxylase and 11 beta-hydroxylase gene expression occur only in adrenal cortex and not in other steroidogenic tissues. A second subclass of tissue-specific expression is the "variable" type whereby 17 alpha-hydroxylase and cholesterol side chain cleavage (SCC) activity are both expressed in ovarian thecal cells but only SCC activity is expressed in corpus luteum. The third type of regulation is cAMP-independent and leads to fetal-imprinting (initial expression of steroid hydroxylase genes during fetal life).

Adrenal Cortex↗
PubMed1985

Regulation of cytochrome P-45011 beta gene expression by adrenocorticotropin.

Recombinant DNA clones corresponding to 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta) have been identified in a bovine adrenocortical cDNA library. These clones, pB11 beta-1 and pB11 beta-2, hybridize to at least three RNA species, 7.2, 6.2, and 4.3 kilobases in length, present in the adrenal cortex. All three RNA species directed cytochrome P-450(11)beta synthesis in an in vitro translation system. Expression of the cytochrome P-450(11)beta gene is tissue specific in that these transcripts were not detected in liver, heart, kidney, or corpus luteum. In cultured bovine adrenocortical cells, adrenocorticotropin (ACTH) or dibutyryl cAMP increased the concentration of cytochrome P-450(11)beta transcripts. This increase appears to be at least partially due to de novo transcription as shown by the action of actinomycin D which effectively blocked the ACTH-induced increase in cytochrome P-450(11)beta RNA level. Furthermore, cycloheximide administered prior to or along with ACTH resulted in the blockage of any new transcription of the cytochrome P-450(11)beta gene as evidenced from the level of RNA. Thus, in addition to potential effects on RNA stabilization, a primary action of ACTH in the regulation of the level of cytochrome P-450(11)beta may be to mediate the induction of an unidentified adrenocortical protein which is required for the activation of cytochrome P-450(11)beta gene expression. These results, together with previous studies (Kramer, R.E., Rainey, W.E., Funkenstein, B., Dee, A., Simpson, E. R., and Waterman, M. R. (1984) J. Biol. Chem. 259, 707-713) that showed increased intracellular levels of cAMP upon treatment of bovine adrenocortical cell cultures with ACTH, suggest a role for cAMP-mediated events in the regulation of cytochrome P-450(11)beta gene expression.

Adrenocorticotropic Hormone↗
PubMed1985

Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide.

Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services--namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11 beta-hydroxylase cytochrome P-450.

Adrenal Cortex↗
PubMed1984

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts approximately equal to 425 and approximately equal to 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species approximately equal to 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.

Adrenal Cortex↗
PubMed1983

Reaction of nitric oxide with heme proteins: studies on metmyoglobin, opossum methemoglobin, and microperoxidase.

Kinetic and EPR studies show that the first step in the reaction of NO with ferric myoglobin, opossum hemoglobin, and microperoxidase is the reversible formation of the H-NO complex: H + NO in equilibrium H-NO (where H = Mb+, or Hb+ OP, or MP+). The NO-combination rates are markedly affected by the presence or absence of the distal histidine. The distal histidine significantly reduces the NO-combination rates, perhaps by interaction between the distal histidine and the ferric iron. Thus the beta-chains of Hb+ OP and metmyoglobin show similar combination rates. In the absence of a distal histidine, the NO-combination rates in the alpha-chains of Hb+ OP are much faster and similar to those observed for the five-coordinate heme in microperoxidase. The loss of a water molecule from the six-coordination site is assumed to be the rate-limiting step.

Animals↗
PubMed1983

[Do repetitive DNA sequences have a biological function?].

By DNA reassociation kinetics it is known that the eucaryotic genome consists of non-repetitive DNA, middle-repetitive DNA and highly repetitive DNA. Whereas the majority of protein-coding genes is located on non-repetitive DNA, repetitive DNA forms a constitutive part of eucaryotic DNA and its amount in most cases equals or even substantially exceeds that of non-repetitive DNA. During the past years a large body of data on repetitive DNA has accumulated and these have prompted speculations ranging from specific roles in the regulation of gene expression to that of a selfish entity with inconsequential functions. The following article summarizes recent findings on structural, transcriptional and evolutionary aspects and, although by no means being proven, some possible biological functions are discussed.

Animals↗