1H, 13C and 15N assignments of the KorA global transcriptional repressor protein from the low copy number IncP-1 plasmid, RK2.
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Amino Acids↗
Biomedical subjects
C M Thomas
Publications and source records attributed to C M Thomas.
Chondrocyte death by apoptosis is associated with cartilage matrix degradation.
OBJECTIVES: To investigate the frequency of chondrocyte apoptosis in equine articular cartilage (AC) specimens and to examine the relationship between the process of cell death and the degree of cartilage degradation using a direct quantification of numbers of apoptotic cells and expression of active caspase-3. METHODS: AC from equine metacarpophalangeal (MCP), proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints was used and each joint was graded macroscopically for cartilage degradation (macroscopic osteoarthritis (OA) score). Cartilage sections were graded using a 'modified' Mankin scoring system. Apoptosis of chondrocytes in cartilage sections was assessed morphologically by appearance of apoptotic features (direct method) and by expression of active caspase-3 using indirect immunohistochemistry. RESULTS: The extent of apoptosis assessed by the direct method did not show any relationship with increasing severity of OA (P=0.72). However, there was a significant positive correlation between 'modified' Mankin score and apoptosis determined by caspase-3, with the extent of apoptosis found to increase linearly with increasing severity of OA (r=0.44, P=0.0043). Caspase-3 expression was found to be significantly higher in the superficial and middle zones than in the deep zone (P<0.001). In the superficial, middle and deep zones, expression of caspase-3 was significantly higher in the MCP joint than in the PIP joint (P=0.013, P=0.0018 and P=0.029, respectively). Within the MCP joints, apoptosis was higher in the lateral compartment compared to the medial (P=0.053). CONCLUSIONS: The data presented in this study demonstrate that chondrocyte apoptosis is positively associated with degree of cartilage matrix damage and that the extent of apoptosis varies with cartilage zones and mechanical loading environment of the joint.
Animals↗
Transcription regulatory circuits in bacterial plasmids.
Gene regulation circuits control all aspects of the life of plasmids. This review gives an overview of the current orchestration of the circuits that control plasmid replication, plasmid transfer, plasmid segregation and plasmid maintenance.
Bacteria↗
Analysis of marker or complex chromosomal rearrangements present in pre- and post-natal karyotypes utilizing a combination of G-banding, spectral karyotyping and fluorescence in situ hybridization.
The significance of complex chromosomal rearrangements presents a diagnostic dilemma. In the past, the use of G-banding coupled with fluorescence in situ hybridization (FISH) has been the standard approach. The recent development of spectral karyotyping (SKY) and multicolor FISH (M-FISH) has resulted in an increased accuracy of identification of marker or other complex chromosomal rearrangements. However, owing to the additional cost and time associated with SKY or M-FISH, and the restricted availability of such imaging facilities in many centers, it is not feasible to perform these procedures routinely on every sample. In addition, the identification of an aberration by SKY or M-FISH will often require confirmation by FISH. A practical approach is needed to take advantage of the complementary strengths of each method. In our center we utilize an algorithm that dictates the use of routine G-banding for the initial preliminary evaluation of a patient, followed by SKY characterization if marker chromosomes or complex translocations are detected by the G-banding analysis. According to this algorithm, FISH is used to verify the results once the origin of the abnormal chromosome has been determined by SKY. To demonstrate the effectiveness of this algorithm, we have analyzed both amniocyte and lymphocyte slides, using a combination of G-banding, SKY, and FISH. Our results confirm that an algorithm which selectively uses SKY or M-FISH will provide an efficient and improved method for pre- and post-natal chromosomal analysis.
Algorithms↗
The 64 508 bp IncP-1beta antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1beta group.
The complete 64508 bp nucleotide sequence of the IncP-1beta antibiotic-resistance plasmid pB10, which was isolated from a waste-water treatment plant in Germany and mediates resistance against the antimicrobial agents amoxicillin, streptomycin, sulfonamides and tetracycline and against mercury ions, was determined and analysed. A typical class 1 integron with completely conserved 5' and 3' segments is inserted between the tra and trb regions. The two mobile gene cassettes of this integron encode a beta-lactamase of the oxacillin-hydrolysing type (Oxa-2) and a gene product of unknown function (OrfE-like), respectively. The pB10-specific gene load present between the replication module (trfA1) and the origin of vegetative replication (oriV) is composed of four class II (Tn3 family) transposable elements: (i). a Tn501-like mercury-resistance (mer) transposon downstream of the trfA1 gene, (ii). a truncated derivative of the widespread streptomycin-resistance transposon Tn5393c, (iii). the insertion sequence element IS1071 and (iv). a Tn1721-like transposon that contains the tetracycline-resistance genes tetA and tetR. A very similar Tn501-like mer transposon is present in the same target site of the IncP-1beta degradative plasmid pJP4 and the IncP-1beta resistance plasmid R906, suggesting that pB10, R906 and pJP4 are derivatives of a common ancestor. Interestingly, large parts of the predicted pB10 restriction map, except for the tetracycline-resistance determinant, are identical to that of R906. It thus appears that plasmid pB10 acquired as many as five resistance genes via three transposons and one integron, which it may rapidly spread among bacterial populations given its high promiscuity. Comparison of the pB10 backbone DNA sequences with those of other sequenced IncP-1beta plasmids reveals a mosaic structure. While the conjugative transfer modules (trb and tra regions) and the replication module are very closely related to the corresponding segments of the IncP-1beta resistance plasmid R751 and even more similar to the IncP-1beta degradative plasmids pTSA and pADP-1, the stable inheritance operons klcAB-korC and kleAEF are most similar to those of the IncP-1beta resistance plasmid pB4, and clearly less similar to the other IncP-1beta plasmids. This suggests that IncP-1beta plasmids can undergo recombination in the environment, which may enhance plasmid diversity and bacterial adaptability.
Anti-Bacterial Agents↗
Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2.
Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon. Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids. The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer. Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively. Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC. For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255. The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218. Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC. This IncC-KorB contact differs in location from the ParA-ParB/SopA-SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant.
Amino Acid Sequence↗
Transposition patterns of unlinked transposed Ds elements from two T-DNA loci on tomato chromosomes 7 and 8.
We have previously reported that unlinked transposed Ds elements originating from chromosome 4 of tomato preferentially inserted in chromosome 2. This observation, together with data from other studies, suggested that there may be absolute preferences for transposition, irrespective of the chromosomal location of the donor site. The aim of the present work was to verify whether the distribution of transposed Ds elements on chromosome 2 was non-random and thus whether, unlike the case in maize, unlinked transpositions in tomato are not distributed randomly. To do this, unlinked acceptor sites of Ds elements originating from two donor T-DNA loci lying on chromosomes 7 and 8 were mapped. Receptor sites for tr Ds elements transposed from the 1601D locus on chromosome 8 exhibited a non-random distribution (P<0.01). Eleven out of 46 independent transpositions mapped to chromosome 2 and, as this was statistically significant (P<0.01), proves that receptor sites for this element are not randomly distribution on the chromosomes. In addition, deviation of the observed number from the expected number of tr Dss was close to being significant for chromosome 4 (P=0.05-0.1). In contrast, the distribution of unlinked receptor sites for tr Dss derived from the 1481J locus on chromosome 7 was random. Chi(2)tests were performed for each chromosome, and for chromosome 4 the difference between the observed and the expected number of tr Dss was very high but statistically non-significant (P=0.05-0.1). For chromosome 2 the difference was statistically negligible. Therefore, we conclude that chromosome 2 does not serve as a preferential receptor for the transposition of Ds elements independently of the location of the donor site.
Base Sequence↗
The bacterial ParA-ParB partitioning proteins.
A pair of genes designated parA and parB are encoded by many low copy number plasmids and bacterial chromosomes. They work with one or more cis-acting sites termed centromere-like sequences to ensure better than random predivisional partitioning of the DNA molecule that encodes them. The centromere-like sequences nucleate binding of ParB and titrate sufficient protein to create foci, which are easily visible by immuno-fluorescence microscopy. These foci normally follow the plasmid or the chromosomal replication oriC complexes. ParA is a membrane-associated ATPase that is essential for this symmetric movement of the ParB foci. In Bacillus subtilis ParA oscillates from end to end of the cell as does MinD of E. coli, a relative of the ParA family. ParA may facilitate ParB movement along the inner surface of the cytoplasmic membrane to encounter and become tethered to the next replication zone. The ATP-bound form of ParA appears to adopt the conformation needed to drive partition. Hydrolysis to create ParA-ADP or free ParA appears to favour a form that is not located at the pole and binds to DNA rather than the partition complex. Definition of the protein domains needed for interaction with membranes and the conformational changes that occur on interaction with ATP/ADP will provide insights into the partitioning mechanism and possible targets for inhibitors of partitioning.
Amino Acid Sequence↗
Accumulation of trifluoroacetate in seasonal wetlands in California.
Trifluoroacetate (TFA, CF3COO-) is a stable and mildly phytotoxic breakdown product of several fluorinated organic compounds including the hydro(chloro)fluorocarbons (HFC/HCFCs) that have largely replaced the stratospheric ozone-depleting chlorofluorocarbons (CFCs). TFA enters aquatic ecosystems primarily through precipitation and has the potential to accumulate in water bodies with little or no outflow to the point where toxic concentrations could be achieved. This study demonstrated that seasonal wetlands lacking outflow concentrated TFA as they evaporated during the dry season. In addition, the TFA within the pools was retained between years, which may result in long-term TFA accumulation. Since plants acquire TFA from their growing media, the plants exposed to high aqueous concentrations of TFA within the pools had elevated TFA concentrations with a median concentration of 279 ng/g dry weight in their tissues as compared to 33 ng/g for species growing outside the pools. The highest TFA concentrations in water, which occurred just prior to the pools drying up, were in the 2-10 micrograms/L range. These concentrations are approximately 190 or less than reported toxic concentrations for the most sensitive species tested, but our evidence suggests that these concentrations will increase with continued TFA deposition into the pools.
Environmental Monitoring↗
Vascular endothelial growth factor in ovarian cyst fluid.
BACKGROUND: The purpose of the current study was to determine vascular endothelial growth factor (VEGF) concentrations in cyst fluid from malignant, borderline, and benign ovarian tumors, and to correlate these data with preoperative serum VEGF concentrations and clinicopathologic characteristics. METHODS: One hundred seven ovarian cysts were removed and punctured for cyst fluid collection. Histologic diagnosis revealed 25 malignant, 12 borderline, and 70 benign ovarian tumors. The VEGF concentrations of all the cyst fluid specimens as well as in 37 preoperatively collected serum samples were determined by making use of a sandwich type double determinant enzyme linked immunoadsorbent assay based on a combination of 4 polyclonal antibodies. RESULTS: Statistically significantly higher VEGF concentrations were found in cyst fluid from malignant (median, 21.5 microg/L) compared with borderline (median, 3.2 microg/L; P = 0.01) or benign tumors (median, 1.3 microg/L; P < 0.0001). Preoperative serum VEGF concentrations were significantly higher in patients with malignant (median, 0.63 microg/L; range, 0.016-17.7 microg/L) compared with nonmalignant tumors (median, 0.28 microg/L; range, 0.016-0.89 microg/L; P = 0.008). A significant correlation of preoperative serum VEGF was found with VEGF cyst fluid concentrations (r = 0.38; P = 0.02). Significantly higher VEGF cyst fluid concentrations were found in serous malignant (median, 31.9 microg/L) compared with mucinous malignant tumors (median, 4.7 microg/L; P = 0.004). Not significant, though higher median VEGF cyst fluid concentrations were found in advanced International Federation of Gynecology and Obstetrics (FIGO) Stage II, III, and IV, histologic Grade 2 and 3, patients with residual tumor greater than 2 cm, with malignant cells in ascites or peritoneal washings, or with recurrent disease, as compared with FIGO Stage I, histologic grade 1, patients with less than or equal to 2-cm residual tumor, without malignant cells in ascites/peritoneal washings, or without recurrent disease, respectively. CONCLUSIONS: It has become clear from the increased study sample that ovarian tumors of different histologic etiology vary in VEGF cyst fluid concentrations, with the highest VEGF cyst fluid concentrations in malignant tumors. The prognostic significance of VEGF cyst fluid concentrations in advanced FIGO stage seems to be of limited value but may be important in the selection of high risk FIGO Stage I and borderline types. Data from this study indicate a possible role for VEGF as a serum tumor marker.
Adenocarcinoma, Mucinous↗
Regulatory circuits for plasmid survival.
Bacterial plasmids deploy a diverse range of regulatory mechanisms to control expression of the functions they need to survive in the host population. Understanding of the mechanisms by which autoregulatory circuits control plasmid survival functions, in particular plasmid replication, has been advanced by recent studies. At a molecular level, structural understanding of how certain antisense RNAs control replication and stability functions is almost complete. Control circuits linking plasmid transfer functions to the status of the bacterial population have been dissected, uncovering a complex and hierarchical organisation. Coordinate or global regulation of plasmid replication, transfer and stable maintenance functions is becoming apparent across a range of plasmid families.
DNA Replication↗
Longitudinal vitamin and homocysteine levels in normal pregnancy.
Evidence of the impact of maternal nutritional status on pregnancy outcome is increasing. However, reference values for vitamin and homocysteine concentrations in maternal blood during normal pregnancy are scarce, and are lacking for the preconceptional period and early pregnancy. Thus, in a longitudinal study we evaluated vitamin and homocysteine concentrations in 102 nulliparous women with an uneventful singleton pregnancy and normal outcome not using supplements. The physiological changes in vitamin and homocysteine concentrations in blood were determined from the preconceptional period throughout pregnancy until 6 weeks post-partum. The vitamins evaluated comprised retinol, thiamin, riboflavin, pyridoxal 5'-phosphate, folate in serum and erythrocytes, vitamin B12 and alpha-tocopherol. The plasma homocysteine concentration was also measured, considering the essential roles of folate, vitamin B6 and vitamin B12 in homocysteine metabolism. The concentrations of retinol, thiamin, pyridoxal 5'-phosphate serum folate and vitamin B12 decreased during pregnancy. In contrast, the concentrations of riboflavin, alpha-tocopherol, and folate in erythrocytes increased or showed only minor changes. Homocysteine concentrations also remained approximately constant during pregnancy. These observations emphasize the importance of preconceptional and post-partum concentrations of vitamins in the evaluation of pregnancy-induced changes. These data have provided valuable reference values for vitamins and homocysteine before, during and after pregnancy in order to contribute to better diagnosis of maternal deficiencies and to study further the relationship between maternal vitamin status and adverse course and outcome of pregnancy.
Case-Control Studies↗
Increased serum FSH in female fragile X premutation carriers with either regular menstrual cycles or on oral contraceptives.
Fragile X premutations are known to be a risk factor for diminished ovarian function at a relatively young age. We studied endocrine profiles of female fragile X family members (n = 79) at risk of premature ovarian failure (POF). Of these 79 women aged <40 years, 45 had menstrual cycles, and 34 were using oral contraceptives. Of the women with menstrual cycles, the premutation carriers had higher serum FSH concentrations than women who were not carrying the premutation. Even premutation carriers with regular cycles showed increased serum FSH concentrations. Moreover, premutation carriers using oral contraceptives also demonstrated increased serum FSH concentrations. Irrespective of whether oral contraceptives were used, a serum FSH concentration of > or =15 IU/l was more common in the premutation carriers than in the other women. One premutation carrier using oral contraceptives had a serum FSH concentration of >40 IU/l, the threshold that defines POF. We confirmed that premutation carriers with menstrual cycles demonstrate premature ovarian dysfunction. However, we also found endocrine signs of unrecognized ovarian dysfunction in premutation carriers using oral contraceptives, despite endocrine alterations by oral contraceptives. Premutation carriers may have a poorer prognosis for future pregnancy, either achieved spontaneously or by assisted reproductive technology. We recommend that premutation carriers should be counselled not to wait too long if they wish to start a family.
Adult↗
Semen parameters, including WHO and strict criteria morphology, in a fertile and subfertile population: an effort towards standardization of in-vivo thresholds.
In this study, the semen analysis results of a fertile population were compared with those from a subfertile population, in order to establish normal cut-off values for the standard semen parameters with the aid of receiver operating characteristic (ROC) curve analysis. The fertile group comprised healthy males (n = 107) without any history of fertility problems, the partners of whom had had a spontaneous pregnancy within one year of unprotected intercourse and were pregnant at the time of the male's inclusion into the study. A total of 103 males from couples attending the infertility clinic, and with an initial sperm count of <20x10(6)/ml were recruited to form the subfertile population. The best discriminating parameter between the two populations was sperm morphology evaluated according to WHO criteria at a cut-off point of 31% normal spermatozoa. The other cut-off values were at 8% for the acrosome index, 45% for motility, and 4% normal spermatozoa for strict criteria. Recalculating the ROC curve cut-off values based on an assumed 50% prevalence of subfertility in an assisted reproductive setting, the cut-off points were reduced to 21% and 3% normal spermatozoa for WHO and strict criteria respectively. For motility, the new cut-off value was at 20% motile spermatozoa, for motility quality at 3.5 (on a scale of 1-6), the acrosome index at 3% normal acrosomes, and the teratozoospermia index at 2.09.
Acrosome↗
Reduced vitamin B12 binding by transcobalamin II increases the risk of neural tube defects.
Periconceptional folic acid supplementation reduces the risk of neural tube defects (NTD). Homocysteine levels are elevated in mothers of NTD children, which may be due to decreased cellular vitamin B12 levels, as vitamin B12 is a cofactor for the methylation of homocysteine. Transcobalamin II (TC II) transports vitamin B12 to the tissues. To examine whether altered plasma transcobalamin levels are a risk factor for NTD, we determined the apo and holo form of TC II and haptocorrin (TCI+TCIII), vitamin B12 and homocysteine concentrations in the plasma of 46 mothers with NTD children, and in 73 female controls. Holo-tc II levels and holo-tc II percentages (holo-tc II/total tc II) in the first quartile of the control distribution were related to a three-fold (OR 2.9, 95%CI 0.9-9.2) and five-fold (OR 5.0, 95%CI 1.3-19.3) risk, respectively, for having a child with NTD, when compared with the last quartile. Homocysteine levels were significantly higher among individuals with low holo-tc II, low total vitamin B12 concentrations and low holo-tc II percentages. These low holo-tc II percentages are probably caused by reduced affinity of TC II for vitamin B12, which may be explained by genetic variation in the TC II gene. Vitamin B12 supplementation might therefore be warranted, in addition to folate, in the prevention of NTD.
Adult↗
Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins.
The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.
Amino Acid Sequence↗
Cooperativity between KorB and TrbA repressors of broad-host-range plasmid RK2.
The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer. trbBp is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the -10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp. Deletion analysis of TrbA showed that the C-terminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.
Amino Acid Sequence↗
Maternal smoking cessation intervention: targeting women and their partners before pregnancy.
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Bias↗