Purification of Class I Medullipins from the Venous Effluent of Isolated Normal Kidneys Perfused under High Pressure with Saline

Medullipin I (Med I) is a vasodepressor prohormone which is continuously elaborated into the renal venous effluent (RVE) of isolated rat kidneys perfused under high pressure. We have improved the yield of Med I by substituting saline for the albumin perfusate previously reported; and considerably improved refinement by directly fractionating the crude lipid extract of the RVE with high pressure liquid chromatography. The results show that Med I, as defined by previous physiologic and pharmacologic criteria, is not a single molecule. The 3 Class I medullipins described here are distinguished by subtle or overt differences in polarity and biologic activity.     

Improved screening for beta-lactam antibiotics. A sensitive, high-throughput assay using DD-carboxypeptidase and a novel chromophore-labeled substrate

The very sensitive and specific method for the detection of beta-lactam antibiotics using DD-carboxypeptidase (DDCase) from Actinomadura strain R39 has been improved to meet the requirements of a high-throughput beta-lactam screening from culture broths of microorganisms. The method is based on a novel chromophor-labeled substrate N alpha-acetyl-N epsilon-4-(7-nitrobenzofurazanyl)-L-lysyl-D-al anyl-D-alanine (ANLA2) which is converted by DDCase into ANLA1 with only one D-alanine residue left. Both compounds are intensely yellow as well as highly fluorescent and can be separated by thin-layer chromatography. This allows easy determination of residual DDCase activity after reaction with beta-lactams by simple visual inspection of chromatograms. Also, many assays can be run at a time without sophisticated instrumentation. Details of the method as well as some results of a beta-lactam screening performed with this type of assay are described.     

Inositol hexaphosphate induces apoptosis by coordinative modulation of P53, Bcl-2 and sequential activation of caspases in 7,12 dimethylbenz[a]anthracene exposed mouse epidermis

Inositol hexaphosphate (IP6) is a major constituent of most cereals, legumes, nuts, oil seeds, and soybean. Anticancer effects of IP6 have been demonstrated in different experimental models. Besides reducing cell proliferation, IP6 increases differentiation of malignant cells, often resulting in restoring the normal phenotype. Exogenously administered IP6 is rapidly taken into the cells and dephosphorylated to lower-phosphate, inositol phosphates, which further interfere with signal transduction pathways and cell cycle arrest. Enhanced immunity and antioxidant properties could also contribute to tumor cell destruction. However, the molecular mechanisms underlying this anticancer action are not fully understood. The present study deals with the effect of topical application of IP6 on some of the selective and critical events of apoptosis in DMBA exposed mouse epidermis. IP6 showed an inhibition of DMBA-induced mutant (mt) p53 expression. Similarly, DMBA induced over expression of Bcl-2 was also reversed by topical treatment of IP6. In addition to the modulation of mt p53 and Bcl-2 expressions, IP6 brought the DMBA-inhibited activity of caspases back to the normal or induced it above the normal levels. The effects of IP6 appeared to be the function of its dose and the duration of its exposure. These results suggested that topically applied IP6 directly induces apoptotic machinery by modulating the expression of mt p53, Bcl-2, and caspase activity.     

Fluorine electron double resonance imaging for 19F MRI in low magnetic fields.

This work demonstrates the feasibility of generating fluorine NMR images at a very low magnetic field of 0.015 T by making use of the Overhauser enhancement of (19)F NMR signal brought about by a stable, water-soluble, narrow-line paramagnetic contrast agent. The enhancement in the (19)F NMR images depends on the concentration of the single electron contrast agent, the pO(2), and the electron paramagnetic resonance (EPR) irradiation power. The applicability of this technique for (19)F NMR imaging is demonstrated with phantom samples, where a time resolution of 4-10 min is achieved. Proton electron double resonance imaging (PEDRI) and fluorine electron double resonance imaging (FEDRI) images were also obtained from rat kidneys ex vivo, perfused with 10 mM Oxo63 and 10 M trifluoroacetic acid. The spatial and temporal resolutions of these images are comparable to those obtained at magnetic fields 2-3 orders of magnitude larger. Constant NMR frequency (628 kHz) operation permits both FEDRI and PEDRI of identical slices without removing the object under investigation. This feasibility of coregistration of proton-based anatomical PEDRI image with physiological FEDRI image offers good potential for studying fluorine-containing tracers.     

Detection of mycobacterium tuberculosis by nested polymerase chain reaction in a case of subconjunctival tuberculosis.

PURPOSE: To highlight the importance of nested polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in a case of subconjunctival tuberculosis. METHODS: We report a case of a 60-year-old man with subconjunctival nodule in the right eye for duration of 6 weeks. Biopsy of the nodule showed a granuloma with extensive caseation necrosis. Ziehl Neelsen staining for acid-fast bacilli (AFB) was negative. However, because of a strong suspicion of Mycobacterium infection, PCR for M. tuberculosis genome was done, using the nested PCR technique. RESULTS: Polymerase chain reaction for M. tuberculosis showed amplification of Mycobacterium tuberculosis genome with the nested PCR technique. CONCLUSION: Our case indicates that PCR can be a valuable tool in the diagnosis of conjunctival tuberculosis from paraffin sections.     

The formation of strand breaks in DNA after high-LET irradiation: a comparison of data from in vitro and cellular systems

This paper presents a summary of our understanding to date of the formation of DNA strand breaks induced by highly energetic particle beams (high-LET radiation). We have compared our own recent data on the formation of strand breaks induced in DNA in an aqueous solution with our previous data and those of others available from the literature for similar lesions made in cellular DNA. When the strand break induction frequency, as number of breaks per Gy per unit DNA, is plotted against LET, a series of biological effect curves (one for each particle atomic number Z) is obtained. The frequency of the formation of single-strand breaks has an RBE of less than 1 for DNA in solution and for DNA in the cell; the frequency of the formation of double-strand breaks (dsb) also has an RBE of less than 1 for DNA in a solution containing low amounts of free radical scavenger(s), while the RBE can be greater than 1 in the 50-200 keV/microns range for cellular DNA. RBE values are with respect to X-rays or cobalt gamma-rays. In cells the level of unrejoined strand breaks is also highest in the 50-200 keV/microns range and may reach 25-35% of the initial break yield depending on particle energy and Z-value. These irreparable lesions include double-strand scissions and some form(s) of single-strand breaks. The data presented cover results obtained for helium to uranium particles, with an LET range of 16 to 160,000 keV/microns. When different biological end-points are compared a strong correlation is found between induction of dsb, chromosomal abnormalities and mutation induction.     

Modulation of carcinogen metabolism and DNA interaction by calcium glucarate in mouse skin.

Almost all the polycyclic aromatic hydrocarbons (PAHs) require metabolic activation to exert their carcinogenic activity. Environmental carcinogen [(3)H] benzo[a]pyrene (BP) is carcinogenic only after its metabolic transformation to a reactive intermediate, which can then bind to cellular macromolecules. Inhibition of dimethylbenz anthracene- (DMBA-) DNA binding generally accompanied inhibition of tumor initiation as most inhibitors of initiation interfere with the metabolic activation of the initiator. The importance of carcinogen-DNA interaction and the enzymes involved in the metabolism of carcinogenic polycyclic hydrocarbons has led to a search for inhibitors that would be useful in modifying the cancer-causing effects of the PAHs. We tested the effect of calcium glucarate (Cag), a naturally occurring nontoxic compound, on carcinogen metabolism and DNA interaction. Cag inhibited [(3)H] BP binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. Application of Cag to mouse skin caused a dose-dependent inhibition of [(3)H] BP binding to epidermal DNA. To establish the relevance of the in vivo results to the in vitro situation, we followed the in vitro effect of Cag on [(3)H] BP binding to calf thymus DNA and observed that Cag inhibited the [(3)H] BP binding to calf thymus DNA in the presence of microsomes prepared from animals treated with DMBA. We also studied related events like DNA synthesis and carcinogen metabolism. For assessing the DNA synthesis, thymidine kinase was used as marker. Cag caused a dose-dependent inhibition of DMBA-induced thymidine kinase activity. At the same time, Cag caused a marked inhibition of DMBA-induced aryl hydrocarbon hydroxylase (AHH) activity, an enzyme responsible for the metabolism of PAHs like BP, both in vivo and in vitro. Our study indicates that Cag exerted its antitumor effect possibly by inhibiting the carcinogen-DNA binding, which appears to be due to reduced DNA synthesis and AHH activity.     

Intracranial pressure in childhood cerebral malaria

Lumbar punctures were performed in 40 Gambian children with acute cerebral malaria aged between 18 months and 10 years. The mean opening pressure was elevated in 32 (80%) of the children, but was not significantly different in the 14 fatal cases compared with survivors: 110 (standard deviation 71) versus 131 (58) mm of cerebrospinal fluid respectively. Cerebral perfusion pressures were also similar in the 2 groups: 64 (20) mm Hg versus 64 (11) mm Hg respectively. There was no clear clinical evidence of raised intracranial pressure, and no evidence of deterioration immediately following lumbar puncture. Nevertheless brain swelling, and consequent brain-stem compression, may contribute to a fatal outcome in cerebral malaria--particularly in those children who die from sudden respiratory arrest. A prospective evaluation of osmotic agents in childhood cerebral malaria seems to be justified.