Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

Long-term chemotherapy of HIV-associated Kaposi's sarcoma with liposomal doxorubicin.

The aim of this study was to examine the outcome, adverse events and clinical complications of long-term chemotherapy with pegylated liposomal doxorubicin (PegLiposomal DOX) for human immunodeficiency virus (HIV)-associated Kaposi's sarcoma (KS) in the pre-highly active antiretroviral therapy (HAART) era. A phase II study over a 4-year period in a tertiary care university hospital was carried out. 52 acquired immunodeficiency syndrome (AIDS)-patients with advanced KS received long-term chemotherapy (71+/-51 weeks) with a mean of 22.8+/-18.2 cycles and a mean cumulative liposomal doxorubicin dose of 456+/-364 mg/m(2) (120-1040 mg/m(2)). Tumour burden, duration and dosage of PegLiposomal DOX, adverse events, opportunistic infections, immunological parameters and HIV load were measured. A complete (10%) or partial response (56%) was achieved while on chemotherapy. 10 patients (19%) showed stable disease. Tumour progression was observed in 8 patients (15%). Importantly, chemotherapy with PegLiposomal DOX was also successful after previous cytostatic therapy with bleomycin and vincristine. The most common adverse events included leucopenia, neutropenia, anaemia, and increased liver function tests. 34 patients (65%) developed new opportunistic infections and 29 patients (56%) died during the study period. To conclude, pegylated liposomal doxorubicin is a safe and effective drug for long-term chemotherapy of advanced (AIDS) KS without adverse effects on CD4 cell counts and HIV viral load.

Three-dimensional structure of fatty-acid-binding protein from bovine heart.

The complex of fatty-acid-binding protein (FABP) from bovine heart (cFABP, pI4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle-shaped crystals belong to the monoclinic space group C2, with unit-cell constants a = 5.262(6) nm, b = 7.631(8) nm, c = 3.945(5) nm and beta = 94.47(9) degrees. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods. An initial model for the three-dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T.A., Bergfors, T., Sedzik, J. & Unge, T. (1988) EMBO J. 7, 1597-1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all-atom and a united-atom force field. The optimized models were used to determine the crystal structure of cFABP by molecular-replacement techniques. The structure was refined by simulated annealing to R = 0.267. As the bound lipid is heterogeneous, it could not be located in the electron-density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel beta strands forming a beta barrel, and by two alpha helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters.

Subcellular distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay.

Several types of the 14-15 kDa fatty acid-binding proteins (FABPs) are known to occur in the cytosol of mammalian cells. With antibodies raised against the cardiac-type protein from bovine heart, immunoblots indicated a more widespread distribution of the cardiac FABP in subcellular fractions, such as mitochondria and nuclei. A detailed view was obtained when the post-embedding protein A-gold labeling method was applied to cross-sections of heart cells and isolated subcellular fractions. Cardiac FABP in myocytes was associated with myofibrils and localized within mitochondria and nuclei. After subfractionation of mitochondria, the binding protein was recovered with matrix proteins only. A non-competitive enzyme-linked immunosorbent assay (ELISA) of the direct type was developed specifically for bovine cardiac FABP. This assay was sensitive in the range of 0.05 to 1 ng, and concentrations of cardiac FABP per mg protein were found for cytosol, matrix and nuclei to be around 3.18, 0.18 and 0.03 micrograms, respectively. The newly found compartmentation of cardiac FABP in the heart cell must be considered when the true functions of the protein, yet to be defined, are studied.

Development of a low-dose monthly injectable contraceptive system: II. Pharmacokinetic and pharmacodynamic studies.

A drug delivery system which provides a sustained release of norethindrone (NET) and mestranol (ME) for one month after a single intramuscular injection was assessed as a long-acting injectable contraceptive. The system is based upon well defined particle size crystals of the synthetic steroids maintained in suspension with saline solution. Eight healthy ovulating women volunteered for the study; they received a combination of 10 mg of NET plus 1 mg of ME in 1 ml of vehicle by intramuscular injection on day five of their menstrual cycle. Blood samples were drawn at 0, 1, 5, 10, 13, 17 and 21 days after drug administration. The immunoreactive serum levels of estradiol, progesterone, NET and ethinylestradiol were measured by specific radioimmunoassay procedures to assess ovarian function and the kinetic parameters of the synthetic steroids. This newly developed contraceptive system proved to be both effective, and long-lasting as well as devoid of side effects.

Development of a low-dose monthly injectable contraceptive system: I. Choice of compounds, dose and administration route.

A low-dose combined estrogen-progestogen sustained release system has been developed. The control of crystal size of the steroids produced sustained dissolution in vitro and in vivo. A dose-response experiment carried out in healthy women indicated that 10 mg of norethisterone (NET) with 1 mg of ethinylestradiol (EE2) when given by intramuscular injection maintained NET serum levels above 1 ng/ml for at least 25 days. The system is considered suitable for one month contraceptive protection.

PIP: This study was aimed at assessing a new low dose combined estrogen-progestogen sustained release system in which the steroids were formulated as a microcrystalline aqueous suspension and injected throug a 19-gauge needle. The control of crystal size of norethisterone (NET) and ethinyl estradiol (EE2) produced sustained dissolution in both in vitro and in vivo studies. In vitro dissolution profiles revealed an inverse relationship between the percentage of NET and EE2 dissolved and size range of crystals, with complete dissolution of the miconized steroid achieved at 1 hour. A dose-response experiment carried out in healthy women indicated that intramuscular injection of 10 mg of NET with 1 mg of EE2 maintained NET serum levels above 1 ng/ml for at least 25 days. Systemic side effects such as nausea and vomiting were experienced by all 7 volunteers after the injection, suggesting that EE2 was rapidly absorbed. A less soluble system such as mestranol could be used instead of EE2 to improve the performance of the present system. There were no signs of local irritation. These findings indicate that the dosages of currently available injectable contraceptive compounds can be substantially reduced without loss of anovulatory potency. Clinical studies to assess the pharmacokinetics and pharmacodynamics of 10 mg of NET with 1 mg of EE2 or mestranol as monthly injectable are currently under way.

The influence of beta blockers on cardiovascular reactivity and Type A behavior pattern in hypertensives.

The influence of beta blockers on Type A behavior pattern and cardiovascular reactivity was tested. Nineteen white, male hypertensive patients were divided at random into two therapeutic groups (100 mg atenolol versus 50 mg hydrochlorothiazide + 5 mg amiloride (control group) ). For each patient, the German version of the structured interview was performed before therapy and at a minimum of 4 weeks after normalization of clinical resting casual blood pressure (BP). Prior to therapy, there were no differences in age, BP at rest, cardiovascular reactivity, and Type A between the two groups. After therapy, the patients treated with beta blockers changed Type A characteristics toward Type B, regardless where they started on the Type A scale, and beta blockers attenuated cardiovascular reactivity. In this study, Type A (hypertensive patients) was not associated with greater cardiovascular reactivity.

Plasma levels and urinary excretion of orally administered propantheline bromide in man.

After a single oral dose of 30 or 60 mg of propantheline bromide peak plasma levels of the drug were reached within 2 h in six healthy men. Mean peak plasma concentrations were 20.6 and 53.1 ng/ml after 30 mg and 60 mg respectively. The mean apparent absorption and elimination half-lives after 30 mg dose were 0.22 and 1.57 h respectively, and similar half-lives were found at the higher dose level. There was a dose related change in plasma levels and AUCinfinity of the drug, and some 3% to 4% of the administered dose of propantheline bromide was excreted unchanged in urine at each dose level. Comparison of the plasma levels and urinary excretion of the drug with those seen after i.v. administration in an earlier study indicated an apparently low systemic availability of orally administered propantheline bromide. There was tentative evidence of a qualitative relationship between the oral dose administered, plasma concentrations and the effects of propantheline bromide on salivary excretion.