PubMed Health⌕ Search

Biomedical subjects

C M Thomas

Publications and source records attributed to C M Thomas.

At least 271 records · Page 15Linked to original sources
PubMed1984

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative bacterial species. To analyze the elements of its control mechanism, we have measured the copy number in Escherichia coli of mini-RK2 replicons isogenic except for defined deletions in regions adjacent to the vegetative replication origin, oriVRK2, which have previously been implicated in copy number control because of their expression of plasmid incompatibility. The results indicate that while the previously defined 700-bp HaeII oriVRK2 fragment carries one copy control element (copA), a second (copB) lies at least partly outside this fragment towards the tetracycline resistance genes of RK2. Deletions affecting both these regions give a mini replicon with a copy number of 35-40 compared with 4-7 for parental RK2. Further incompatibility experiments indicate that targets for both incA (copA) and incB (copB) lie within the 700-bp HaeII oriVRK2 fragment.

Base Sequence↗
PubMed1984

The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression.

For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity. The trfA gene is transcribed as part of a polycistronic operon which also includes kilD. Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci. Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself. Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter. This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans. When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2. Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis. These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication.

Bacterial Proteins↗
PubMed1984

Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB.

Transcription at various points in the trf A region of broad host range plasmid RK2 has been analysed by measuring expression of the galK gene inserted at EcoRI sites introduced previously by TB1723 transposition mutagenesis. Rightward transcription (anti-clockwise on RK2) probably from a single promoter, proceeds across two open reading frames coding for a 13 kD polypeptide of unknown function, and the trf A gene, which provides a protein(s) essential for plasmid replication. This transcription is not auto-regulated by the products of either open reading frame and is also not subject to significant attenuation prior to the end of the trfA open reading frame. Leftward transcription appears to be directed by at least two well separated promoters, the more leftward being three to four times stronger than the more rightward. Rightward, but not leftward, transcription is repressed about 9-fold by the trfB locus of RK2 alone (so far not separable from the loci korA and korD) in trans while the combination of the korB and trfB loci in trans represses both rightward transcription (about 100-fold) and leftward transcription (the stronger activity by 10 to 15-fold). Regulation of these operons is therefore qualitatively different. The kilD locus in the trfA region, which is suppressed by korD (trfB) is thus probably part of the rightward (trfA) operon, while leftward transcription may represent the start of an operon containing kilB. The results suggest that RK2kor loci act by repressing transcription of kil loci and that the kil and kor control circuits may be part of an interlocking system of RK2 genes involved in replication and stable maintenance.

Chromosome Mapping↗
PubMed1984

Hormonal characteristics of women with clinical features of the polycystic ovary syndrome.

The aim of the present study was to determine whether a group of patients selected on the basis of clinical features only is characterized by the typical hormonal findings as discussed in the literature concerning the PCO-syndrome. PCO patients had oligomenorrhea, secondary amenorrhea or otherwise evidence of chronic anovulation, as well as hirsutism and/or obesity. Control women had regular menstrual cycles and a normal body weight. Since androgen and estrogen production in women depends on the stage of follicular development, an effort was made to obtain endocrinological data under standardized conditions. Under well-defined circumstances the PCO group (n = 20) had higher LH levels and lower FSH levels as compared with the control group (n = 10). Consequently the LH/FSH ratio was significantly elevated in the PCO group. Serum estrone and estradiol levels were significantly elevated in the PCO group, as were the serum levels of androstenedione and testosterone. Despite these differences a marked degree of overlap existed in the PCO patients and the control women for gonadotropin, estrogen and androgen levels. It was concluded that although the presence of polycystic ovaries in the investigated PCO group of women was not confirmed by laparoscopy, laparotomy or histological examination of the ovaries, these women had basal endocrinological characteristics similar to those found in well-proven PCO patients reported in the literature.

Adult↗
PubMed1984

Pituitary and ovarian responses to LHRH stimulation in women with clinical features of the polycystic ovary syndrome.

The gonadotrophin release and the changes in ovarian and adrenal steroid levels following the administration of a low dose of LHRH were assessed in a group of women with clinical features of the polycystic ovary syndrome (PCO syndrome). The results were compared with those obtained in a group of normal ovulatory women. A significant increase in LH and FSH levels and a decrease in cortisol concentrations were demonstrated in both study groups following the administration of LHRH. The LH response was significantly exaggerated in the PCO group when compared with the control women. The estrone and estradiol levels did not change in either group. The androstenedione and testosterone concentrations did not change or decreased in the control group, whereas an increase of these hormones was seen in the PCO group. In the PCO group a positive correlation between the LH response and the androstenedione response was noticed. These findings indicate that the hyperandrogenic state encountered in PCO patients is at least in part of ovarian origin.

Adult↗
PubMed1984

Pituitary gonadotropin responses to different modes and doses of intravenous luteinizing hormone-releasing hormone administration in normal men.

In 22 potentially fertile men, pituitary gonadotropin secretion was investigated by intravenous luteinizing hormone-releasing hormone (LH-RH) administration. LH-RH was administered continuously (1 microgram/minute) and in a pulsatile fashion (20 micrograms at 20-minute intervals, 20 micrograms at 60-minute intervals, and 60 micrograms at 60-minute intervals), for 3 hours, under standardized conditions. Blood was collected continuously by means of an integrated sampling technique. The mean serum luteinizing hormone (LH) concentration after any type of pulsatile administration rose significantly more than after continuous LH-RH administration. The mean increase in LH after pulsatile LH-RH administration with a 60-micrograms dose and 60-minute intervals was significantly greater than after pulsatile administration with a 20-micrograms dose and 20- or 60-minute intervals. No differences were observed in follicle-stimulating hormone responses after any type of LH-RH administration. These data confirm the existence of a self-priming effect for LH in the men; and maximum pituitary stimulation, within the dosage range tested, is reached after pulsatile LH-RH stimulation with an interval of 60 minutes.

Adult↗
PubMed1984

Differential effect of luteinizing hormone-releasing hormone infusion on testicular steroids in normal men and patients with idiopathic oligospermia.

Basal serum gonadotropin levels in 11 oligospermic men were significantly higher than in 9 euspermic control subjects, although most were still in the normal range. Basal serum testosterone (T), 17-hydroxyprogesterone (17-OHP), and estradiol levels and their ratios did not differ significantly. Continuous luteinizing hormone-releasing hormone (LH-RH) infusion (1 microgram/minute for 180 minutes) during integrated blood sampling evoked similar gonadotropin responses in both groups but had a differential effect on T: in the control subjects T increased (P less than 0.01) within 15 minutes to 1.5 times baseline, whereas in the oligospermic men T decreased (P less than 0.01). From 60 minutes on, however, T also significantly rose in the oligospermic men, but the maximum increment was about half lower (P less than 0.01) than in the euspermic men, despite virtually similar rises in 17-OHP. Only in the oligospermic men did the 17-OHP/T ratio increase (P less than 0.02) during LH-RH, which is compatible with the occurrence of a 17,20-lyase block. Serum estradiol did not increase in either group. In conclusion, continuous LH-RH infusion uncovers an intrinsic difference in acute Leydig cell stimulation between euspermic and oligospermic men.

Adult↗
PubMed1984

Genetic evidence for the direction of transcription of the trfA gene of broad host range plasmid RK2.

For replication of broad host range plasmid RK2 in Escherichia coli two regions of the plasmid genome are essential, oriV RK2 between 12.0 and 12.7 kb on the genome (defined clockwise from the unique EcoRI site) and trfA, located between 16.0 and 17.4 kb, which provides a trans-acting product necessary for oriV RK2 function. The properties of an insertion mutant of a mini-RK2/ColE1 hybrid plasmid suggest that the trfA promoter lies clockwise from the 17.4 kb RK2 coordinate. Fusion of the trfA gene lacking its normal promoter to the E. coli trpE gene in a hybrid plasmid confirmed that trfA is transcribed anticlockwise, towards the Tcr gene of RK2. Repression of trpE promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of trfA gene expression. The results also indicate the presence of a transcription unit running anticlockwise through the trfB region located between 54.0 and 56.0 kb on the RK2 genome.

Escherichia coli↗
PubMed1984

Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2.

A 3.2 kb region of the broad host range IncP plasmid RK2 (indistinguishable from RP1, RP4, R68 and R18) anticlockwise from the EcoRI site may be separated phenotypically into three loci. The trfB/korA/korD locus both complements a temperature-sensitive maintenance mutation and suppresses the deleterious effects of the loci kilA and kilD; the incC locus expresses incompatibility towards complete RK2-like replicons, and the korB locus suppresses the host lethal effect the kilB locus. Transcriptional fusions of the galK gene to various segments of this region revealed that all three loci are transcribed anticlockwise from a common promoter. A weak secondary promoter may also contribute to the expression of korB. Analysis of the sizes of the polypeptides produced from these segments led to the identification of two cistrons, the first encoding a polypeptide of 38 kDal associated with incC function and the second a polypeptide of 49 kDal associated with korB activity. The trfB/korA/korD activities are associated with a polypeptide of 14kDal which may be an N-terminal fragment of the incC-associated polypeptide.

Chromosome Mapping↗
PubMed1984

Intracerebral hemorrhage secondary to cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is a cause of intracerebral hemorrhage in a significant proportion of normotensive patients. Two cases of pathologically proved multiple intracerebral hemorrhages due to CAA are reported. These hemorrhages are accurately demonstrated on computed tomography and are typically superficial in location. CAA should be considered as a cause of such hemorrhages in elderly patients who are often normotensive and demented.

Aged↗
PubMed1984

Distinct role of neonatal and adult monocytes in the regulation of the in vitro antigen-induced plaque-forming cell response in man.

Mononuclear cells from human cord blood (CBMC) are able to mount an antigen-specific IgM plaque-forming cell (PFC) response after primary in vitro stimulation with the T cell-dependent antigen ovalbumin (OA). The antigen dose-response relationship for the induction of PFC in cultures of CBMC is represented by a bell-shaped curve comparable to that found for mononuclear cells from adult peripheral blood (adult PBMC). The dose of OA optimal for the induction of a response in cultures of CBMC consistently, however, is 100-fold lower than the antigen dose optimal for adult PBMC (0.03 microgram OA/ml vs 3.0 micrograms OA/ml). Results obtained from co-culture experiments in which semiallogeneic combinations of parental/neonatal lymphocytes and monocytes were stimulated with a variable dose of OA indicate that the adherent cell (AC) plays a pivotal role in the establishment of the optimum antigen dose. From experiments using antigen-pulsed AC, it was concluded that neonatal and adult AC differ in their antigen handling capacity. In the presence of the prostaglandin synthetase inhibitor indomethacin the antigen dose-response relationship for the induction of PFC in cultures of CBMC shifts to an "adult type" of curve. From pulsing experiments it emerges that indomethacin affects the interaction between antigen and monocytes. Indomethacin causes an enhancement of the expression of HLA-DR at the surface of neonatal as well as adult AC; this can be down regulated by the addition of prostaglandin E2 (PGE2). The addition of PGE2 to cultures of adult PBMC leads to a shift of the optimal antigen dose for induction of PFC toward lower concentrations. Although higher levels of PGE2 were measured in the supernatant of cultured neonatal AC compared with adult AC, it seems unlikely that this observation can explain the distinct antigen dose-response relationship for the induction of a PFC response in cultures of CBMC.

Adult↗
PubMed1983

Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2.

Figurski et al. (1982) have reported that certain loci on the broad host range plasmid RK2 (kil functions) can be cloned only in the presence of other trans-acting segments of the plasmid genome (kor functions). They have suggested that the presence of these functions may in part account for the structure of mini RK2 replicons which were constructed in order to define the regions of the plasmid which encode replication/maintenance functions (Thomas et al. 1980). We have therefore investigated the relationship between these two sets of kil and kor loci and the loci implicated in the replication/maintenance of RK2. We find that, whilst the three kil loci reported by Figurski et al. (1982) are absent from these derivatives, a fourth such locus (kilD) is closely linked to trfA, a gene essential for RK2 replication. The kilD locus was probably responsible for the inclusion in mini replicons of a segment of RK2 DNA which carries both korD and korA in addition to trfB, a gene defined by a temperature-sensitive maintenance defect, but which can be deleted leaving a functional RK2 replicon (Thomas 1981 b). The kilB locus is situated on the opposite side of kilD from trfA, all three loci lying within a 3.6 kb segment of RK2 DNA. The korA, korD and trfB functions all map within a 900 bp segment of DNA, while korB requires sequence information at least 1.5 kb from this segment.

DNA Replication↗
PubMed1983

Instability of a high-copy-number mutant of a miniplasmid derived from broad host range IncP plasmid RK2.

Mini-RK2 plasmids pCT460 and pCT461 which contain the oriVRK2, trfA and trfB regions of RK2 in addition to tetracycline and kanamycin resistance determinants, have copy numbers of 17 and 35 copies per chromosome equivalent, respectively. The difference in copy number is due to a 56-bp deletion in oriVRK2 in pCT461. In Escherichia coli only pCT461 is markedly unstable in batch culture while both are unstable (although pCT461 is more so) in bacteria on stock plates. The instability of pCT461 in bacteria on stock plates is recA+ dependent and appears to involve loss of plasmid DNA from bacteria rather than selective cell death. After storage of recA+ bacteria carrying pCT461 for a few weeks the remaining antibiotic-resistant bacteria carry a mixture of plasmid DNA species including parental pCT461, transposable element insertion derivatives, and, by far the majority, deletion derivatives. It appears that one particular plasmid region, which includes the kilD gene (which inhibits plasmid maintenance in the absence of korD which, however, is present on pCT460 and pCT461), is responsible for this instability in a gene dosage-dependent way. Most of these deletion derivatives are dependent on pCT461-specified trfA gene (essential for replication) so that they do not displace pCT461 entirely. Their presence reduces the copy number of pCT461, thus reducing the instability, and is probably ultimately responsible for pCT461 survival on stock plates. In many bacteria the same process which gives rise to deletion derivatives may result in degradation of plasmid DNA extensive enough to cause loss of pCT461.

Chromosome Deletion↗
PubMed1982

Variation among normal persons in short-term ventilatory capacity.

Among individuals of comparable size with normal lungs, short-term breathing capacity varies widely for unknown reasons. If the velocity of ventilatory muscle contraction limits airflow, then persons with a high proportion of fast twitch fibers in the ventilatory muscles should have large ventilatory capacities. As one test of this hypothesis, we correlated ventilatory capacity with vertical jump, an index of leg and thus of diaphragmatic muscle fiber composition. We found significant positive correlations between vertical jump and the 12-second maximal voluntary ventilation in both women (n = 19, r = 0.6; p less than 0.01) and men (n = 25; r = + 0.4; p = 0.06). Both correlations were independent of body weight or vital capacity. These results are consistent with the hypothesis that short-term ventilatory capacity is influenced by the contractile characteristics of the ventilatory muscles.

Adult↗
PubMed1981

Urodynamic studies in the normal menstrual cycle: the relationship between hormonal changes during the menstrual cycle and the urethral pressure profile.

The influence of endogenous gonadal steroids on the urethral pressure profile (UPP) was studied in 27 healthy nulliparous women with the normal ovulatory cycles. The UPP and hormonal values were recorded in the follicular phase, at midcycle, and early and late in the luteal phase. Simultaneous urethrocystometry was performed by means of the microtransducer technique, according to a precise standardized methodology. The parameters of the UPP, as defined by the Standardization Committee of the International Continence Society, and the area under the urethral closure pressure curve (integrated pressure) were calculated. Reproducibility of the UPP measurements was evaluated. In addition to a basal body temperature (BBT) chart, serum levels of 17 beta-estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin were measured at each study session in order to confirm a normal ovulatory pattern for that cycle and for comparison with the urodynamic parameters. The mean values and standard deviation of the UPP parameters in healthy nulliparous women in the supine and sitting positions are presented. Values of the pressure measurements obtained during one menstrual cycle did not change systemically with the hormonal alterations. At midcycle and early in the luteal phase, however, and anatomic and functional lengths of the urethra were consistently increased over those found in the early follicular and late luteal phases. The data suggest a causal relationship between the changes in serum E2 concentrations and the changes in urethral length.

Adult↗