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Biomedical subjects

A Barkan

Publications and source records attributed to A Barkan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid.

Results of in vitro experiments have suggested the existence of at least three pathways by which nuclear-encoded proteins are targeted to the chloroplast thylakoid membrane. However, few components of the targeting machinery have been identified and the relationship between the three pathways is not clear. To investigate mechanisms underlying thylakoid protein targeting, we identified nuclear mutations in maize that cause targeting defects. We found two mutations, tha1 and hcf106, that disrupt the localization of different sets of proteins to the thylakoid lumen. The tha1 mutation interferes with the targeting of one chloroplast-encoded protein, cytochrome f, and three nuclear-encoded proteins, plastocyanin, the psaF gene product and the 33 kDa subunit of the oxygen-evolving complex. The hcf106 mutation interferes with the targeting of the 16 and 23 kDa subunits of the oxygen-evolving complex. The tha1 and hcf106 phenotypes provide the first in vivo evidence supporting the existence of two distinct thylakoid-targeting pathways. Their phenotypes also provide evidence that one chloroplast-encoded protein, cytochrome f, engages the 'tha1' pathway, indicating that nuclear- and chloroplast-encoded proteins can be targeted via common machinery.

Adenosine Triphosphatases↗
PubMed1994

A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms.

A mutant designated crp1 (chloroplast RNA processing 1) was identified in a screen for transposon-induced maize mutants with defects in chloroplast gene expression. crp1 is a recessive, nuclear mutation that causes the loss of the cytochrome f/b6 complex and a reduction in photosystem I. The molecular basis for these protein losses is unique relative to previously described mutants with defects in organelle gene expression; it involves defects in the metabolism of two organellar mRNAs and in the translation of two organellar proteins. Mutants lack the monocistronic forms of the petB and petD mRNAs (encoding cytochrome f/b6 subunits), but contain normal levels of their polycistronic precursors. Pulse-labeling experiments revealed normal synthesis of the petB gene product, but a large decrease in synthesis of the petD gene product. These results suggest that petD sequences are more efficiently translated in a monocistronic than in a polycistronic context, thereby providing evidence that the elaborate RNA processing typical of chloroplast transcripts can play a role in controlling gene expression. Structural predictions suggest that the petD start codon lies in a stable hairpin in the polycistronic RNA, but remains unpaired in the monocistronic transcript. Thus, processing to a monocistronic form may increase translational efficiency by releasing the translation initiation region from inhibitory interactions with upstream RNA sequences. Synthesis of a third cytochrome f/b6 subunit, encoded by the petA gene, was undetectable in crp1, although its mRNA appeared unaltered. Two mechanisms are consistent with the simultaneous loss of both petA and petD protein synthesis: the translation of the petA and petD mRNAs might be coupled via a mechanism independent of crp1, or the crp1 gene may function to coordinate the expression of the two genes, which encode subunits of the same complex.

Alternative Splicing↗
PubMed1993

L-thyroxine malabsorption due to the injection of herbal remedies.

Two patients are described in whom the absorption of l-thyroxine was impaired by non-prescription herbal and nutritional remedies. The absorption of thyroid hormones is discussed and an approach to the problem of patients who appear to be unresponsive to the usual doses of thyroid hormones is suggested.

Absorption↗
PubMed1992

Fasting-induced changes in thyroid hormone economy in normal and acromegalic subjects.

To study whether high growth hormone (GH) milieu may counteract fasting-induced changes in thyroid hormone economy, we measured basal and TRH-stimulated TSH concentrations as well as thyroxine (total, TT4 and free, FT4), total triiodothyronine (TT3) and total reverse T3 (TrT3) before and after a 6-day fast in 6 healthy men and in 8 patients with acromegaly. Baseline values for all parameters were similar in both groups. Fasting induced similar increases in TT4 and TrT3 concentrations and a similar decline in TT3 concentrations in both groups. The TT3/TT4 and TrT3/TT4 ratios changed identically in both groups. We conclude that high GH is incapable of altering fasting-induced changes in thyroid hormone economy.

Acromegaly↗
PubMed1991

Inactivation of maize transposon Mu suppresses a mutant phenotype by activating an outward-reading promoter near the end of Mu1.

We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon. When the Mu transposon system is in an active phase, hcf106 conditions a nonphotosynthetic, pale green phenotype. However, when the Mu system is inactive (a state correlated with hypermethylation of Mu elements), the plant adopts a normal phenotype despite the continued presence of the transposon within the gene. The molecular mechanisms that mediate this suppression of the mutant phenotype have now been investigated. We show here that the Mu element responsible for the hcf106 lesion lies within sequences encoding the 5'-untranslated leader of the Hcf106 mRNA. When the Mu transposon system is active, this insertion interferes with the accumulation of mRNA from the hcf106 allele. However, when Mu is inactive, mRNA similar in size and abundance to that transcribed from the normal allele accumulates. These transcripts initiate at many sites throughout a 70-base-pair region, within and immediately downstream of the Mu1 insertion. Thus, an unusual promoter spanning the downstream junction between Mu1 and Hcf106 substitutes for the normal Hcf106 promoter but only when Mu is inactive. The pattern of mRNA accumulation in different organs and in response to light suggests that the activity of this promoter is conditional not only upon the phase of Mu activity, but also upon signals that regulate the normal Hcf106 promoter.

Base Sequence↗
PubMed1991

Dexamethasone alters responses of pituitary gonadotropin-releasing hormone (GnRH) receptors, gonadotropin subunit messenger ribonucleic acids, and gonadotropins to pulsatile GnRH in male rats.

Dexamethasone (Dex), when administered in high doses, has been shown to suppress spontaneous and GnRH-induced gonadotropin secretion, but the level and the mechanism(s) of this effect are unknown. We administered Dex to castrate testosterone-replaced male rats to determine if gonadotropin gene expression is affected and whether Dex differentially influences GnRH-modulated parameters of gonadotrope function: induction of GnRH receptors (GnRH-R) and gonadotropin synthesis and secretion. GnRH was given iv at 25 ng/pulse at 8, 30, and 120 min intervals for 48 h. Rapid GnRH injection frequency preferentially increased alpha and LH-beta messenger RNA (mRNA) responses to GnRH as well as LH secretion. Slower GnRH injection frequencies were required to increase levels of GnRH-R, FSH-beta mRNA, and FSH secretion. Dex selectively inhibited the serum LH, alpha, and LH-beta mRNA responses to GnRH, but not the serum FSH or FSH-beta mRNA responses. Additionally, it augmented the GnRH-induced increase in GnRH-R. We conclude: 1) induction of GnRH-R, gonadotropin synthesis, and secretion require different modes of GnRH stimulation; 2) Dex acts directly on the gonadotrope to differentially modulate GnRH-induced increases in GnRH-R levels, gonadotropin gene expression, and gonadotropin secretion; and 3) GnRH effects upon induction of GnRH-R, LH, and FSH synthesis and secretion are likely to be mediated via different cellular pathways.

Animals↗
PubMed1990

Somatically heritable switches in the DNA modification of Mu transposable elements monitored with a suppressible mutant in maize.

Many transposable elements in maize alternate between active and inactive phases associated with the modification of their DNA. Elements in an inactive phase lose their ability to transpose, their ability to excise from reporter alleles and, in some cases, their ability to enhance or suppress mutant phenotypes caused by their insertion. The maize mutant hcf106 is a recessive pale green seedling lethal caused by the insertion of the transposable element Mu1. We show that the hcf106 mutant phenotype is suppressed in lines that have lost Mu activity. That is, homozygous hcf106 seedlings are dark green and viable when transposable elements belonging to the Robertson's Mutator family are modified in their terminal inverted repeats, a diagnostic feature of inactive lines. This property of the mutant phenotype has been used to follow clonal leaf sectors containing modified Mu elements that arise from single somatic cells during plant development. The distribution of these sectors indicates that epigenetic switches involving Mu DNA modification occur progressively as the meristem ages.

Blotting, Southern↗
PubMed1989

Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator.

The maize photosynthetic mutant hcf106 has a distinctive and unusual thylakoid membrane organization, and fails to accumulate three of the five thylakoid membrane protein complexes. This mutant arose in a Robertson's Mutator background, and shows somatic instability typical of a transposon-induced allele. In addition, hcf106 is suppressed when Mu1 elements are inactive and modified in their terminal inverted repeats. Thus plants homozygous for the mutant allele adopt a mutant phenotype only when Mu1 elements are active and unmodified. DNA from the mutant allele has been cloned by 'transposon-tagging' using the transposon Mu1, and the identity of the clone confirmed by observing somatic excision of the transposon in a revertant sector. A 1.2 kb transcript homologous to the cloned DNA is found in wild-type and suppressed seedlings, but is not found in mutant seedlings, suggesting that suppression is mediated at the level of transcript accumulation.

Alleles↗
PubMed1989

Tissue-dependent plastid RNA splicing in maize: transcripts from four plastid genes are predominantly unspliced in leaf meristems and roots.

Most plastid gene products do not accumulate to high levels in meristem proplastids or in the specialized plastids of roots. To assess whether a modulation of plastid splicing activities might play a role in this tissue-dependent expression of the plastid genome, the ratio of spliced to unspliced transcripts from the atpF, petB, petD, and rpl16 genes was compared between several tissues of maize. Although these transcripts are predominantly spliced in green leaf tissue (both bundle sheath and mesophyll cells), spliced atpF, petB, and petD transcripts are underrepresented relative to their unspliced precursors in roots and leaf meristems. The ratio of spliced to unspliced rpl16 transcripts varies in a similar fashion, but the magnitude of the differences between tissues is not as great. The proportion of RNA that is spliced reflects the tissue of origin and not photosynthetic competency, chlorophyll content, or exposure to light since the leaves of photosynthetic mutants and of seedlings grown in the absence of light contain spliced and unspliced transcripts in normal ratios. These results raise the possibility that low RNA splicing activities are in part responsible for the limited expression of the plastid genome in meristematic and root tissue.

Gene Expression Regulation↗
PubMed1988

Acromegalic arthropathy. Characteristics and response to therapy.

Arthropathy was assessed in 19 patients with active acromegaly. Axial or peripheral arthropathy was present in 10 patients, was located most often in large joints or the lumbosacral spine, and was osteoarthritic in nature. The mean duration of acromegaly in patients with arthropathy was 21.6 years, while the mean duration in patients without arthropathy was 7.9 years. A mild-to-moderate improvement in symptoms, estimated functional ability, and crepitus occurred in 8 of 9 patients who were prospectively examined during therapy to lower production of growth hormone. We conclude that this therapy did improve the symptoms of acromegalic arthropathy. Whether objective structural improvements occur remains unclear.

Acromegaly↗
PubMed1988

Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs.

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of overlapping RNAs through a series of processing steps. The functional significance of this complicated mode of expression is unknown. To determine whether processing of the primary transcript is required to create translatable mRNAs, the translational properties of the RNAs derived from the maize psbB gene cluster (containing the psbB, psbH, petB and petD genes) were examined. Almost all of the approximately 20 RNAs derived from this region co-sediment with polysomes in sucrose gradients, suggesting that at least one coding region on most transcripts is translated. To determine which sequences are translated on each polycistronic RNA, antibodies to psbB, petB or petD proteins were used to immunoselect polysomes engaged in the synthesis of each protein. Northern and S1 nuclease analyses of the immunoselected RNAs revealed that (i) potential start codons within the petB and petD introns are not functional in translation; (ii) all transcripts containing spliced petB or petD sequences are translated to give these proteins, regardless of upstream or downstream sequences; (iii) psbB is translated from all transcripts encoding it. It is concluded that intercistronic processing is not required for translation of these RNAs, although certain processing steps may enhance translational efficiency.

Blotting, Northern↗
PubMed1988

Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40.

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.

Animals↗
PubMed1987

Use of nuclear mutants in the analysis of chloroplast development.

Although a wide range of mutations in the nuclear genome also affect chloroplast biogenesis, their pleiotropic nature often limits their use in studying nuclear genes that regulate or facilitate chloroplast development. However, many mutations that cause a high-chlorophyll-fluorescent (hcf) phenotype exhibit limited pleiotrophy, causing the loss of functionally related sets of chloroplast polypeptides. Several hcf mutations are described that result in the loss of one specific protein complex from the thylakoid membrane. Chloroplast and cytosolic mRNAs coding for component polypeptides of the missing complex are unaffected in the mutants, suggesting that each mutation disrupts some process in the synthesis and assembly of the missing complex. Another hcf mutation causes both the loss of three protein complexes and grossly abnormal thylakoid membrane structures. The primary effect of this mutation might be in the assembly of thylakoid membranes or in the stable accumulation of the three protein complexes. Two other hcf mutations are more pleiotropic. Hcf*-38 causes a quantitative reduction of many chloroplast proteins and a reduction of some chloroplast RNAs, including several splicing intermediates. Hcf*-7 causes a major reduction of all chloroplast-encoded proteins examined. The range of pleiotropic effects of hcf mutations indicates that the mutations identify nuclear genes whose products are involved in a number of different steps in chloroplast development. Because some of the mutations described have been generated by transposon insertions, they can be cloned using the transposon to identify the mutant allele.

Cell Nucleus↗
PubMed1987

The maize plastid psbB-psbF-petB-petD gene cluster: spliced and unspliced petB and petD RNAs encode alternative products.

The chloroplast psbB, psbF, petB, and petD genes are cotranscribed and give rise to many overlapping RNAs. The mechanism and significance of this mode of expression are of interest, particularly because the accumulation of the psb and pet gene products respond differently to both light and, in C4 species such as maize, developmental signals. We present an analysis of the maize psbB, psbF, petB, and petD genes and intergenic regions. The genes are organized similarly in maize (a C4 species) and in several C3 species. Functional class II-like introns interrupt the 5' ends of petB and petD. Both spliced and unspliced RNAs accumulate; these encode alternative forms of the petB and petD proteins, differing at their N-termini. Promoter-like elements between psbF and petB, and biased codon usage suggest that the differential regulation of the psb and pet genes might be achieved at both the transcriptional and translational levels.

Amino Acid Sequence↗
PubMed1987

Steroid biosynthesis in human adrenal tumors.

Patients with adrenal tumors present with varied clinical features which may be related to differing patterns of adrenal steroidogenesis. To explore the mechanism underlying these differences, we studied the in vitro activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17-hydroxylase (17-OH), 21-hydroxylase (21-OH), and 17-20 desmolase (17, 20-D) in 6 adrenal tumors, 4 adenomas and 2 carcinomas. Normal human adrenal tissue was also studied for comparison. Adrenal adenomas had increased 21-OH activity compared with normal adrenal tissue (11.4 +/- 0.7 vs 5.5 +/- 0.5 nmol/mg prot/min, P less than 0.002) and with adrenal carcinomas (11.4 +/- 0.7 vs 3.3 +/- 0.9 nmol/mg prot/min, P less than 0.001). Carcinomas had reduced 3 beta-HSD, 17-OH and 17,20-D activities when compared to controls, but this did not reach statistical significance. These observations suggest less efficient steroidogenesis by adrenal carcinomas, a finding which may explain the large size of these tumors when the symptoms of hypercortisolism first appear.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Gonadotrophin responses to GnRH pulses in hypogonadotrophic hypogonadism: LH responsiveness is maintained in the presence of luteal phase concentrations of oestrogen and progesterone.

LH pulse secretion changes during the menstrual cycle from a rapid regular pattern in the follicular phase to a slower and irregular pattern in the luteal phase. To determine whether the irregular LH pulse pattern in the luteal phase reflects altered GnRH secretion or altered pituitary responsiveness to GnRH, we gave low dose GnRH pulses (25 ng/kg i.v.) every 2 h or every hour for 10 or 12 d to three women with isolated GnRH deficiency. After 4 d of GnRH alone, oestradiol (E2) was given and after 6 d progesterone (P) was added to mimic the hormonal milieu of the luteal phase. LH and FSH were measured every 4 h throughout and also every 20 min for 6 or 12 h, before and after GnRH alone (day 0 and day 4), after E2 (day 6), and after E2 + P (day 10 and day 12). Both GnRH pulse frequencies resulted in a rapid increase in plasma FSH to peaks on day 4 (every 2 h) and day 2 and 3 (every hour). FSH concentrations then declined as plasma E2 rose to 50-80 pg/ml reflecting the selective inhibitory effect of E2 on FSH release. Plasma LH was also increased after the hourly GnRH injections and this regimen was associated with a more rapid rise in E2 reflecting follicular maturation. In contrast to the differences in mean hormone concentrations, administration of GnRH at both frequencies resulted in sustained one-on-one responsiveness of LH that was maintained in the presence of both oestrogen and progesterone at mid-luteal phase concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗