Expression-level dependent activation of recombinant human parathyroid hormone/parathyroid hormone-related peptide receptor: effect of human parathyroid hormone (1-34), (1-31), and (28-48).

A stable recombinant chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10(5) to I.9 x 10(6) receptors per cell. The affinity of the receptors for hPTH-(1-34) was independent of the receptor number per cell (Kd approximately = 8 nmol/1). The induction of cAMP by hPTH-(1-34) is maximal in clones expressing >2x10(5) receptors per cell and Ca++ signals were maximal in cell lines expressing >1.4x10(6) receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca++ are independent and Ca++ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(1-31) showed also an increase in intracellular Ca++. Even in cell lines expressing more than 10(6) receptors per cell the Ca++/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.

[Burnout syndrome in women in surgery]. / Das Burnout-Syndrom bei Frauen in der Chirurgie.

During the past years the burnout syndrome has internationally gained importance especially within the medical professions. With the help of a questionnaire it was investigated at German clinics in how far the Burnout Syndrome occurs among female surgical interns, leading to the result that 68 percent of the poll participants have shown burnout. Reasons that lead to this burnout were among other things doing more than nine hours of overtime per week, the feeling of psychic and physical burdenings during operations as well as disappointed anticipation and unsuitable strategies to assimilate burdenings.

Heat-labile uracil-DNA glycosylase: purification and characterization.

A uracil-DNA glycosylase (UNG) from a psychrophilic marine bacterium (BMTU 3346) has been purified to apparent homogeneity. The enzyme has a molecular weight of 23400 Da. It is stable in complex buffers (containing glycerol/BSA), whereas it is heat-labile in dilute buffers (free of stabilizers) with a half-life of 2 min at 40 degrees C. Due to the thermolability, uracil-DNA glycosylase is suitable for application in the carryover prevention technique showing less residual activity and/or a slower reactivation rate than the usually applied UNG from Escherichia coli.

MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'.

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).

McrI: a novel class-II restriction endonuclease from Micrococcus cryophilus recognizing 5'-CGRY/CG-3'.

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.

Long-term effects of a complex behavioral treatment of essential hypertension.

A multidimensional behavioral program was developed for the treatment of essential hypertension. Over a course of 6 weeks, 108 patients of a rehabilitation center were treated with this program consisting of health education, self-monitoring of blood pressure, relaxation techniques, and a social skill training in addition to standard medical treatment alone. Blood pressure and other cardiovascular risk factors were monitored for a period of 12 months. Blood pressure levels became normotensive in both groups at the end of the 6-week training program. However, the blood pressure reduction in the behavioral treatment group was achieved with fewer patients taking antihypertensive medication than in the control group. The number of patients taking antihypertensive drugs in the treatment group was 60.2% prior to treatment and 44.4% after treatment; figures for the control group were 68% and 73.8%, respectively (p less than or equal to 0.01). Almost identical data were obtained at the 6- and 23-month follow-up examinations. No consistent changes were observed in weight, smoking, or blood lipids. These results demonstrate beneficial and long-lasting effects of a combined medical and psychologic treatment of essential hypertension as compared to medical treatment alone.

Activation of the Bradyrhizobium japonicum nifH and nifDK operons is dependent on promoter-upstream DNA sequences.

Previous analysis of B. japonicum nifH'- and nifD'-'lacZ translational fusions showed that these promoters could be activated by the K. pneumoniae nifA plus the E. coli ntrA gene products. To study the functions of the DNA 5' to these promoters, plasmids carrying deletions in this region were constructed and analyzed in vivo in a heterologous system consisting of an E. coli (NtrA+) background with a plasmid that constitutively expresses the K. pneumoniae nifA gene. Activation of the B. japonicum promoters was completely dependent on sequences located between positions -165 and -100, relative to the start of transcription. Some of the nifD deletion-fusions were mobilized to the wild-type B. japonicum and the exconjugants tested in an ex planta micro-aerobic system, and also used to infect soybean seedlings. The time course of derepression was followed by assaying beta-galactosidase activity from samples withdrawn from the microaerobic cultures or from root-nodule extracts. The results conclusively show that in the homologous system the sequences upstream of the promoter are required to achieve wild-type activity.

Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome.

Two different repeated sequences (RSs) were discovered in the Rhizobium japonicum genome: RSRj alpha is 1126 base pairs long and is repeated 12 times; RSRj beta is approximately 950 base pairs long and is repeated at least 6 times. Their arrangement in root nodule bacteroid DNA is the same as in DNA from bacteria grown in culture. Deletion analysis showed that many copies of alpha and beta are clustered around the nitrogenase genes nifDK and nifH, or, in general, they are found within a genomic region harboring genes that are nonessential for growth. One copy each of alpha and beta are located upstream of nifDK and are adjacent to each other. Neither of them, however, is involved in the expression of nifDK. Nucleotide sequence analysis of three copies of RS alpha revealed many characteristics of procaryotic insertion sequence elements: potential inverted repeats at their ends, potential target site duplication, and large open reading frames. Despite this, their genomic positions appear to be stable. One possible function of these RSs is in deletion formation probably via recombination between them.

In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated.

In contrast to Klebsiella pneumoniae or fast-growing Rhizobium species, such as R. meliloti, where the nitrogenase structural genes are clustered in one operon (nifHDK), in slow-growing Rhizobium japonicum 110, nifH and nifDK are on separate operons.