Laser desorption ionization-mass spectrometric (LDI-MS) analysis of vital biological tissues and native, ex vivo tissue specimens is described. It was found that LDI-MS analysis yields tissue specific data using lasers both in the ultraviolet and far-infrared wavelength regimes, while visible and near IR lasers did not produce informative MS data. LDI mass spectra feature predominantly phospholipid-type molecular ions both in positive and negative ion modes, similar to other desorption ionization methods. Spectra were practically identical to rapid evaporative ionization MS (REIMS) spectra of corresponding tissues, indicating a similar ion formation mechanism. LDI-MS analysis of intact tissues was characterized in detail. The effect of laser fluence on the spectral characteristics (intensity and pattern) was investigated in the case of both continuous wave and pulsed lasers at various wavelengths. Since lasers are utilized in various fields of surgery, a surgical laser system was combined with a mass spectrometer in order to develop an intraoperative tissue identification device. A surgical CO2 laser was found to yield sufficiently high ion current during normal use. The principal component analysis-based real-time data analysis method was developed for the quasi real-time identification of mass spectra. Performance of the system was demonstrated in the case of various malignant tumors of the gastrointestinal tract.

Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I–V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.

BackgroundPhysical training has been shown to improve exercise capabilities in patients with asthma. Most studies focused on children and younger adults. Previously, the maximum program duration was six months. It is not known whether the same results may be obtained with lower intensity programs and sustained for time periods longer than 6 months. This controlled study was undertaken to investigate the effects of a moderate intensity outpatient training program of one year duration on physical fitness and quality of life in adults with asthma.Methods21 adult asthmatics (mean age 56 ± 10 years) were allocated to outpatient training (n = 13) or standard care (n = 8). Exercise consisted of once weekly, 60-minute sessions of moderate intensity. Assessments at baseline and after one year included cardiopulmonary exercise testing and Short Form-36 and Asthma Quality of Life Questionnaires.ResultsFollowing one year of exercise, relevant improvements were observed in the training group for maximum work capacity (p = 0.005), peak oxygen uptake (p < 0.005), O2pulse (p < 0.05), maximum ventilation (p < 0.005), and most of the quality of life domains. No changes were observed in the control group.ConclusionsA physiotherapist-led, long-term, moderate-intensity exercise program of one year duration can induce clinically relevant improvements in exercise capabilities and health-related quality of life in well-motivated adults with asthma.Trial registrationclinicaltrials.gov NCT01097473. Date trial registered: 31.03.2010.

Malonic acid as well as its model substrate methylmalonic acid are covalently bound to two chemically different sites of the fatty acid synthetase complex of yeast. When radioactively labeled malonyl CoA or methylmalonyl CoA are incubated with the multienzyme complex, radioactive malonyl and methylmalonyl enzymes are formed and may be precipitated with trichloroacetic acid. Treatment with performic acid splits only a minor proportion of the (methyl) malonyl enzyme bonds. Most of them are resistant to the oxidant and, hence, prove to be non-thioester linkages.Methy1-[3-W]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methy1)malonyl peptides were separated into three Werent fractions A, B and C. I n peptides B and C (methy1)malonic acid is bound by performic acid stable linkages, whereas (methy1)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor satellite of peptide B, the quantitative relation of both varying somewhat in different preparations. It is concluded that both are derived from a common original peptide sequence in the enzyme.Methyl-[3-14C]malonyI peptide B was further purified by the following procedures : Dowex-BOX2 chromatography, subtilisin S digestion, DEAE-Sephadex chromatography, Dowex-50 X 2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B, end B,, resulted from subtilisin S treatment. They h l l y were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: Bl = Leu,Ala,Gly,Ser,His and B, = Leu, Ah,Gly(S x),Glu,Ser,Hk. Leucine was the C-terminal amino acid in peptide Bl. No cysteine was found in either peptide.A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAESephadex, Dowex-50 X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl peptide B,.The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methy1)malonic acid in peptides B and C is bound to serine by an 0-ester linkage which is unusually activated by some surrounding amino acid, possibly histidine.[3-14c]Malonal peptide A was shown to contain stoichiometric amounts of cysteamine, 8-alanine, and organic phosphate, corresponding to the quantity of malonate bound. These data suggest a thioester linkage between malonate and the SH-group of enzyme-bound 4'-phosphopantetheine. 4'-Phospho-pantetheine, therefore, represents the so called "central" SH-group in yeast fatty acid synthetase. The other malonyl binding site is thought to be the active oenter of the malonyl transferase in the complex.After alkali treatment of the purified fatty acid synthetase under defined conditions 4'-phosphopantetheine was isolated from the hydrolysate and characterized as S-benzoyl-4'-phosphopantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules ...

Connexin 43 (Cx 43)--expressed by germ cells (GC), Sertoli cells (SC) and Leydig cells--is one of at least eleven Cx in the murine testis. A general knockout (KO) of Cx 43 in mice results in perinatal death and a SC-specific KO of Cx 43 (SCCx43KO) causes infertility of male mice by preventing the initiation of spermatogenesis. To further elucidate the role of Cx 43 in the testis, a new mouse model with a GC-specific KO of Cx 43 (GCCx43KO) was created by using the Cre/loxP recombination system. A transgenic mouse line expressing the Cre gene under the tissue non-specific alkaline phosphatase promoter and a transgenic floxed Cx 43-LacZ mouse line were mated. The resulting F1-generation was backcrossed with homozygous Cx 43 floxed mice, and offspring was genotyped. Immunohistochemical analysis of testes of different aged homozygous mice revealed normal spermatogenesis and reduced Cx 43 immunoreactions. RT-qPCR and Western blots showed a downregulation of Cx 43 mRNA and protein, and a nearly unchanged mRNA expression of Cx 26, Cx 33 and Cx 45 in pubertal and adult KO mice. Western blots revealed considerable immunoreactive bands for Cx 26 and Cx 45. Male and female homozygous GCCx43KO mice were viable and fertile. Our data suggest, in contrast to inter SC and inter SC-GC cross talk in SCCx43KO mice which depends selectively on Cx 43 expression, that Cx 43 in GC seems not to be essential in GC-SC communication, when other Cx persist to be expressed.