Deletion analysis identifies a region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which is required for ADR1-mediated derepression.

Deletion analysis was used to identify sequences upstream of the ADH2 gene of Saccharomyces cerevisiae that are required for its regulation. 5' and 3' internal deletions of the ADH2 control region were created in vitro, and the fragments were ligated adjacent to the ADH1 promoter and structural gene. Hybrid genes with 3' deletions extending from -119 to -216 (the start site of ADH2 transcription is designated +1) were fully repressed and derepressed to high levels. Hybrid genes with 3' deletions extending from -119 to -257 were repressed but failed to significantly derepress. Hybrid genes lacking the -216 to -257 region also failed to respond to ADR1-5c, a mutant allele of the unlinked regulatory gene ADR1, which confers constitutive expression on ADH2. This implies that the region between these deletion endpoints, which includes a 22-base-pair sequence of dyad symmetry, is required for efficient derepression of an adjacent promoter. Internal deletions extending in the 3' direction from position -1141 confirmed these results. Deletion mutants lacking the region -1141 to -259 were normally regulated, whereas deletions extending from -1141 to -115 were not derepressible. These results support the hypotheses that the ADH2 promoter may normally be in an inactive conformation in the yeast chromosome and that derepression of ADH2 requires positive activation mediated through an upstream activation sequence located between 216 and 257 base pairs 5' to the start site of ADH2 transcription. No evidence for a DNA sequence mediating repression was obtained.

Construction of multicopy yeast plasmids with regulated centromere function.

The promoter region from the cloned glucose-repressible alcohol dehydrogenase II (ADH2) gene has been inserted in front of the centromere III (CEN3) sequence. It has been shown that, in plasmids containing ADH2-CEN3 fusion (YCRp plasmids), the CEN3 function can be regulated by glucose repression. When yeast transformed with YCRp plasmid are grown in the presence of glucose, the ADH2 promoter is repressed and CEN3 functions normally, mitotically stabilizing the YCRp plasmid. When the carbon source in the culture medium is substituted with ethanol, the transcription from the derepressed ADH2 promoter interferes with the CEN3 function, rendering the YCRp plasmids unstable. At that stage YCRp2 plasmids may accumulate to about ten copies per cell whereas YCRp3 copy number increases to about 100. This demonstrates that the CEN3 sequence can be present on high copy number plasmids in yeast with no detectable toxic effects.

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae.

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.

Lipid peroxidation and scavenging enzyme activity in streptozotocin-induced diabetes.

The aim of this study was to evaluate lipid peroxidation and scavenging enzyme activity in streptozotocin-induced diabetes, and then to establish whether moderate doses of nonenzymatic antioxidant vitamin E play a role in the antioxidant defence system in diabetic pregnant rats and their offspring. The study group consisted of 30 normal female Wistar rats, which were given a single dose of streptozotocin (40 mg/kg) and were mated 7 days later. Subsequently, the diabetic animals were divided into two matched groups: the first supplemented with vitamin E (30 mg/100 g chow), and the other fed with a standard diet lacking vitamin E. Controls consisted of 15 pregnant rats. On the first day after delivery, the rats were decapitated and homogenates of maternal liver and uterus as well as neonatal lungs and liver were prepared. Then the following parameters were measured: malondialdehyde (MDA) concentrations in the homogenates and blood serum, glutathione (GSH) levels, the activity of CuZn-superoxide dismutase (SOD) and glutathione peroxidase (GPx), and glycaemia. The neonates of diabetic rats were smaller than the healthy ones and serum glucose concentration was markedly higher in the diabetic animals. MDA levels were significantly increased, whereas GSH, SOD and GPx were markedly diminished in the diabetic adult rats and their offspring in comparison to the control group. In the animals supplemented with alpha-tocopherol, MDA concentrations were significantly lower, GSH content and SOD activities were markedly elevated most tissues studied, whereas GPx remained unchanged. We conclude that, by monitoring the activity of selected scavenging enzymes, information on ongoing biological oxidative stress and thereby on the fetus/neonate status may be obtained. Our results suggest that diabetic pregnant rats and their neonates are exposed to an increased oxidative stress and that vitamin E supplementation may reduce its detrimental effects.

Antioxidant therapy and streptozotocin-induced diabetes in pregnant rats.

The aim of our study was to analyse the effect of chronic hyperglycaemia on lipid peroxidation and scavenging enzyme activity in pregnant animals and their offspring supplemented and not supplemented with vitamin E - a natural antioxidant. Thirty pregnant female Wistar rats were used in our experiments. Diabetes was induced on day 7 of pregnancy using a single does of streptozotocin (40 mg/kg). Diabetic animals were divided into two equal groups: vitamin E supplemented and those fed with standard diet. Our controls consisted of 15 healthy rats. On day 1 after delivery homogenates of maternal liver and uterus as well as neonatal lungs and liver were prepared. Then the following parameters were measured: malondialdehyde (MDA) concentrations in the homogenates and blood serum, glutathione (GSH) levels, the activity of CuZn superoxide dismutase (SOD) and glutathione peroxidase (GPx) (Bioxytech, France). Statistical analysis was performed using Mann-Withney U test. The neonates of diabetic rats were smaller than those from healthy rats and serum glucose concentration was markedly higher in diabetic animals, both in mothers and neonates. MDA levels increased significantly, whereas GSH content and SOD as well as GPx activities were markedly diminished in diabetic pregnant rats and their offspring in comparison with the control group. In animals supplemented with tocopherol, MDA concentrations declined significantly, GSH contents and SOD activities were markedly elevated in almost all types of tissues studied, whereas glutathione peroxidase remained suppressed. Our results suggest that diabetic pregnant rats and their neonates are exposed to oxidative stress (OS), but vitamin E supplementation could in part reduce the imbalance between uncontrolled reactive oxygen species generation and scavenging enzyme activity, and may potentially serve as a useful prophylactic factor against OS development:

Post-partum evaluation of amylin in lean patients with gestational diabetes mellitus.

The aim of our study was to compare the secretion of amylin, as well as glucose, insulin and C-peptide at baseline and in response to glucagon stimulation in 26 lean women with gestational diabetes mellitus (GDM) and in 19 age- and BMI-matched pregnant women with normal glucose tolerance (NGT). Intravenous 1-mg glucagon stimulation test was performed 6 weeks after delivery. Fasting and stimulated glucose levels were significantly higher in GDM patients than in subjects with NGT ( p<0.01 at 0 and 6 min; glucose area under the curve (AUC), 604.8+/-41.8 mg/6 min vs. 572.4+/-52.4 mg/6 min, p<0.05). Insulin AUC was also markedly higher in GDM subjects than in healthy controls (373.9+/-144.2 micro IU/6 min vs. 283.7+/-139.1 micro IU/6 min, p<0.05). There was no difference in fasting C-peptide levels between the groups studied, but stimulated concentrations, as well as C-peptide AUC were significantly higher in patients with GDM ( p<0.01 at 1 min and p<0.005 at 6 min; AUC, 27.4+/-11.3 pmol/6 min vs. 18.4+/-6.9 pmol/6 min, p<0.01). Amylin levels were higher in GDM group in comparison to healthy subjects ( p<0.005 at 1 and 6 min; amylin AUC, 113.3+/-51.2 pg/6 min vs. 72.5+/-15.7 pg/6 min; p=0.14), but in contrast to the other hormones, did not rise in response to glucagon injection. In conclusion, our results provide evidence that in patients with GDM in the post-partum period, the levels of amylin, as well as the secretion of insulin and C-peptide remain elevated, when compared to women with NTG. Further investigations are needed to clarify the significance of this elevation as a predictive factor for the development of late maternal type 2 diabetes.

Umbilical cord gamma-glutamyl transpeptidase and placental dysfunction.

Placental dysfunction has been suspected if human placental lactogen level and/or cystine-aminopeptidase activity were lower than 10th centile. Significant rise in gamma-glutamyl transpeptidase (GGTP) has been found in samples of arterial cord blood of newborns born to mothers with placental insufficiency. We observed the relationship between GGTP activity and decrease in cord pH and lower Apgar score of corresponding infants. We suggest that chronic hypoxemia as a consequence of placental dysfunction may result in damage of fetal liver cells.

[Strategy in gestational diabetes]. / Strategia postepowania w cukrzycy ciezarnych.

This paper reviews current literature on strategic policy in gestational diabetes (GDM). Emphasis is placed on early identification of carbohydrate intolerance. Definitive guidelines related to screening procedures, diagnostic criteria, glucose monitoring with threshold for insulin therapy in GDM were briefly described. Pregnancy termination and delivery management have been discussed. The importance of postpartum follow-up is pointed out. Special attention is paid to the association with increased antenatal risk of subsequent diabetes and consequently raised perinatal morbidity and/or mortality. For appropriate health-care a cooperation between patients, obstetricians and diabetologists is suggested.

[Familial translocation t (9;16) in a patient with irregular menstrual cycles]. / Rodzinna translokacja t (9;16) u pacjentki z nieregularnymi cyklami miesiaczkowymi.

In this report we present a family with identified carriers of unique reciprocal translocation t (9; 16) (q31; q13) detected through karyotyping of the patient with irregularity of menstrual cycles. Genetic risk for birth of a child with congenital anomalies was estimated as low (0.6%). However, risk for abortion was high. We suggest introducing cytogenetic studies in such cases.

[Analysis of selected methods for intrauterine stimulation of fetal pulmonary maturation]. / Analiza wybranych metod wewnatrzmacicznej stymulacji dojrzewania ukladu oddechowego u plodu.

Preterm delivery is still a major cause of infant morbidity and mortality. Premature infants develop numerous complications including respiratory distress syndrome, intraventricular hemorrhage, patent ductus arteriosus, necrotising enterocolitis, etc. Respiratory distress syndrome is the leading problem in perinatology. The incidence of RDS accounts for 70% of all infants delivered before 30 week's gestation and about 20% neonates born between 32-37 week of pregnancy. Our paper presents current opinions and is a critical review of relevant literature on antenatal treatment before anticipated preterm deliveries. Recently performed meta-analysis prove that glucocorticoid therapy is effective in reducing aforementioned pathologies of prematurity. After brief account on effects of several hormones on fetal pulmonary maturity some beneficial interactions for enhanced fetal maturity are pointed out. Glucocorticoid mechanism of action in target cells and its biochemical implications are reviewed. The use of antenatal corticoids for fetal maturation and possible adverse maternal effects follow. Finally both short-term and long-term benefits of ANS are discussed. Thus there are convincing data to support the use of ANS in fetal pulmonary maturation. The current findings suggest that the improvement of respiratory outcome may depend on enhanced expression of phospholipids and proteins of the surfactant system and enzymes of antioxidant systems. We believe that use of antenatal corticosteroids for fetal maturation results in improvement of neonatal outcome and yields substantial savings in health care costs.

[Intrauterine stimulation for fetal respiratory system maturation; benefits and risks]. / Wewnatrzmaciczna stymulacja dojrzewania ukladu oddechowego; korzysci a ryzyko.

The use of perinatal steroid therapy, first introduced in 1972 is effective in precocious maturation of human lungs. Antenatal corticosteroid therapy results in reduction of fetal mortality, respiratory distress syndrome, intraventricular hemorrhage in preterm babies. These benefits extend to a broad range of gestational age. They comprise the interval between 24 and 34 weeks of human pregnancy and are not limited by the infant's gender or race. The beneficial effects of corticosteroids are the best pronounced after more than 24 hours from the beginning of the treatment. Noteworthy is that therapy less than 24 hours of duration may also improve outcomes. In the presence of premature rupture of membranes, or better with intact membranes, antenatal corticosteroids reduce frequency of RDS, IVH and finally mortality and morbidity. Review of meta-analyses based on randomized trials supports general option that premature infants whose mothers received corticosteroids before delivery are less likely to develop RDS and its complications. Recent data showed that benefits derived from ANS are additive to those of surfactant therapy, rendering the latter more effective. Followup of children up to 12 years of age indicate that ANS do not impair physical growth or psychomotor development. Short-term adverse effects including maternal infection, maternal pulmonary edema were not clearly demonstrated. Pulmonary edema has not been reported when ANS were used alone (i.e. not in combination with betamimetic tocolytics). No long-term unwanted effects on maternal adrenal function have been observed. There is no serious maternal risk resulting from immunosuppressive effect of corticosteroid therapy on maternal immune system. Although glucocorticoid therapy is likely to provoke insulin resistance, and thereby deterioration in diabetic control, and potentially causes cortisol resistance in the fetal lung, the results of scarce randomized trials are not conclusive. In any rate strict control of maternal diabetes mellitus reduces incidence of RDS. Current available data are not indicative of higher risk of fetal mortality in association with maternal hypertensive disease and ANS. In conclusion, most randomized trials of ANS has provided a positive evidence of efficacy and safety of this highly cost effective therapy in most common clinical situations. However, further trials and more precise estimates are justified on ANS treatment specifically related to blood glucose control and evidence concerning the promotion of fetal lung maturity in babies of women with diabetes mellitus. Although benefits of the corticosteroid therapy are beyond any doubts, more experience is needed to assess the effect of ANS on maternal and/or fetal infection in presence of premature rupture of membranes. And finally, further assessments are required on antenatal corticosteroids with dose regimens in patients with multifetal gestation, more common after wide use of techniques of the assisted human reproduction.

[Evaluation of lipid peroxidation and acid-base status in cord blood of newborns after diabetes in pregnancy]. / Ocena peroksydacji lipidów i równowaga kwasowo-zasadowa u noworodka z ciazy powiklanej cukrzyca.

The purpose of our study was to evaluate lipid peroxidation products and scavenging enzyme activity in placenta and cord blood as well as the estimation of acid-base status and blood gases. Seventy five pregnant patients and their newborns were investigated. Twenty eight had pre-gestational diabetes mellitus (PGDM) and 19 gestational diabetes mellitus (GDM). The following parameters were measured: malondialdehyde (MDA) concentrations, glutathione (GSH) levels, the activity of CuZn dismutase (SOD) (Bioxytech, France). Base excess, pO2, pCO2 and pH were measured in arterial and venous samples. Statistical analysis was performed using Mann-Whitney U test. MDA levels and GSH content increased significantly, while SOD activities declined in diabetic group. Newborns of PDGM mothers had essentially diminished pH and rised both, pCO2 and base deficit. There were no any significant differences in parameters of acid-base balance in newborns of patients with GDM as compared with healthy patients. Our results suggest, that in diabetic patients the fetuses are exposed to increased oxidative stress. The evaluation of antioxidant defence and lipid peroxidation, apart from routine measurement of acid-base balance, might serve as a useful marker of fetal distress in diabetic patients.

[Diabetic patient and reproduction].

Current views and opinions on pregnancy in diabetic mothers are presented. Special attention is paid to carbohydrate metabolism, pregestational diabetes mellitus (PGDM), gestational diabetes mellitus (GDM) and unclear heterogenous etiology of gestational diabetes mellitus (GDM). Suggestions for identification, diagnosis and treatment of GDM with an emphasis on early screening strategy are given. Obstetrical management and possible perinatal complications are discussed.

Hemoprotein formation in yeast. VI. Mutants with changed levels of catalase and of other heme enzymes under conditions of glucose repression.

Three mutants with altered response of the hemoprotein level to glucose repression and anoxia were characterized biochemically. Two mutants show complete resistance of catalase activity to glucose repression and a lower degree of resistance of cytochrome c oxidase and of some other enzymes. Liquid nitrogen spectra of these mutants grown in high glucose medium are typical for those of glusoce-derepressed spectra of wild-type cells. The third mutant is hypersensitive to glucose repression and shows only traces of catalase T activity when grown in high-glucose media. In these conditions its spectrum is almost devoid of typical peaks of cytochromes. When grown on ethanol or raffinose it forms catalase and shows a typical derepressed spectrum of cytochromes. The regulatory mechanism impaired in the mutants is not known. It seems likely that the regulation of the heme pathway in the mutants does not differ from that of the wild-type.

Chromatin conformational changes accompany transcriptional activation of a glucose-repressed gene in Saccharomyces cerevisiae.

In the present study we have analyzed the kinetics of DNase I digestion of the two alcohol dehydrogenase (ADH) genes within yeast nuclei. We have found that in yeast grown on glucose the constitutively transcribed ADCI gene is much more sensitive to DNase I digestion than is the repressible ADR2 gene. In yeast grown on ethanol, both genes are transcribed and both exhibit the same sensitivity to DNase I attack. We have also found and mapped DNase I hypersensitive sites near the 5' ends of constitutive and repressible ADH genes. These sites are well correlated with the position at which transcription is initiated.

[Gene therapy perspectives in cystic fibrosis]. / Perspektywy zastosowania terapii genowej w mukowiscydozie.

Cystic fibrosis (CF) is a frequent autosomal recessive genetic disease. The isolation of the gene at the CF locus assigned to the long arm of chromosome 7 band q 31 and defining description of its protein named CFTR (cystic fibrosis transmembrane conductance regulator) promoted understanding the basic biochemical defect. Brief review of relevant literature demonstrates that glycoprotein CFTR is a chloride channel and is activated by a combination of phosphorylation by protein kinase A and binding of ATP. Most common mutation of CF gene, a deletion of the three nucleotides encoding phenylalanine (Delta F508) results in disturbance of chloride transport through membrane of epithelial cells involved in pathomechanism of CF. The way for gene therapy in CF is open, however therapeutic progress is noted on both pharmacologic arena and on the gene cure front. Recombinant vectors utilizing the adenovirus system with high efficiency of CFTR gene transfer to airway epithelium demonstrated in a rat model look promising. The use of retroviruses for CFTR transfer is also advanced mode of somatic gene therapy. An alternative approach suggesting the use of germ line cells is prerequisite of the development of the preimplantation/preconception genetic CF diagnosis. A number of safety and efficacy issues have to be addressed for all approaches before human trials can be implemented.

Cord blood serum creatine kinase isoenzymes with placental dysfunction.

It has been suggested that perinatal asphyxia is not generally followed by neurological impairment unless there is preexisting chronic fetal distress. In cases of brain damage one can observe elevated levels of CK-BB. The purpose of our study was to evaluate CK isoenzymes in umbilical cord blood sera of newborns affected by chronic fetal distress. Fetal distress reflected by placental dysfunction was characterized by a diminished HPL level and decreased activity of CAP. We estimated CK isoenzymes with the use of DEAE-sepharose CL-6B column chromatography. Total CK activity was measured using kits supplied by Boehringer-Mannheim (Monotest CK-NAC aktiviert). The clinical state of examined newborns was estimated. Investigations were carried out in the group of 57 infants delivered after 37 weeks of gestation. Total CK activity in cord sera ranged from 40 to 400 U/l. Our results showed a significant rise of CK-BB activity in cord sera of newborns delivered from pregnancies with placental dysfunction (figure 2) as well as in cases of asphyxiated infants (figure 3). We were unable to demonstrate differences in total CK, CK-MM and CK-MB activities in all examined groups of newborns. Other authors have confirmed that severe asphyxia results in increase in CK-BB activity in cord blood. Infants with ominous fetal heart rate patterns have higher CK-BB activity. There are several possible sources for CK-BB activity in umbilical cord blood sera, i.e. fetal brain, lung, gastrointestinal tract, placenta and uterus. It appears that the brain is most likely the source of elevated CK-BB activity found in cord blood in cases of placental dysfunction.