Perfusion method for preparing pig brain cortex for Golgi-Cox impregnation

The perfusion procedure described in this paper produces high quality impregnation of pig visual and somatosensory cortical neurons with a Golgi-Cox solution. Starting within 30 min after death, pig heads were perfused with a fixative solution composed of a mixture (v/v) of liquid phenol, 5%; formalin, 14%; ethylene glycol, 25%; methanol, 28%; and water, 28% for two periods of 4 hr each. After perfusion, the heads were chilled for at least 18 hr. The entire brain was removed from the skull and then placed in 10% buffered formalin, where it remained for at least 10 days before taking the blocks that were to be immersed in the Golgi-Cox solution. Three weeks spent in the Golgi-Cox solution typically produced uniform neuron impregnation. The tissue blocks were then embedded in celloidin and sectioned at 120 micron. This procedure avoids the following difficulties: Golgi-Cox methods that produced excellent results with rodent or primate tissue were unsuccessful with pig tissue, placing fresh tissue in Golgi-Cox solution resulted in incomplete neuron impregnation, and immersion fixation in 10% buffered formalin without perfusion resulted in excessive staining of glia.     

Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination.

Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13-induced telomeric DNA damage in proliferating cells. Checkpoint-deficient cdc13-1 cells accumulated DNA damage and eventually senesced. However, these telomerase-proficient cells could survive by using homologous recombination but, contrary to telomerase-deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13-1 mec3, as well as in telomerase-deficient cdc13-1 cells, which were Rad52- and Rad50-dependent but Rad51-independent, exclusively amplified the TG(1-3) repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13-1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13-1- Ten1-Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase.     

Wealth and pastoral dairy production: A case study from Maasailand

Much of pastoral development in Africa has been predicted on the assumed desirability of converting pastoralists to commercial beef producers. Such development has ignored two fundamental aspects of pastoral production: first, the greater human support capacity of a dual milk/meat production system, and second, significant wealth inequality within pastoral communities. This paper presents a case study based on several years of field research in Kenya Maasailand; it examines variations by wealth status in milking strategies and the level of milk offtake for human consumption. Rich households have five times the number of cattle per reference adult as poor households, but similar levels of milk consumption, due to differences in the allocation of milk between calves and people. Residential location and watering frequency also vary by wealth status, contributing to a difference in total milk production per cow.The research and the major part of the writing of this paper were done while the author was a scientist with the Kenya Country Program of the International Livestock Center for Africa. Finishing touches were done at the International Laboratory for Research on Animal Disseases. Neither organization bears responsibility for the content. I wish to thank P. N. deLeeuw for long and valuable discussions; the animal science aspects of the research also benefitted from discussions with K. Wagenaar and M. Nicholson. P. Lembuya was, as always, a faithful and demanding assistant. The useful comments of S. Sandford, T. Conelly, U. Herren, and J. Ensminger are also appreciated.     

A hypothesis on p34cdc2 sequestration based on the existence of Ca(2+)-coordinated changes in H+ and MPF activities during Xenopus egg activation [corrected]

The entry into, and exit from, mitosis are controlled by a universal M-phase promoting factor (MPF) composed of at least p34cdc2 and a cyclin. Embryonic systems are convenient for studying the association and dissociation of the active MPF complex because oocytes and eggs are naturally arrested at a specific point of the cell cycle until progression to the next point is triggered by a hormonal signal or sperm. In amphibians, eggs prior to fertilization are arrested at metaphase 2 of meiosis due to the presence of a stabilized MPF complex. Fertilization (egg activation) produces a transient increase in intracellular free Ca2+, a propagating Ca2+ wave, that specifically triggers the destruction of cyclin, leading to MPF inactivation and entry into the first embryonic inter-phase. We have recently shown that intracellular pH (pHi) variations in amphibian eggs, a large increase at fertilization and small oscillations during the embryonic cell cycle, were temporally and functionally related to the corresponding changes in MPF activity. In addition, the recent finding that the pHi increase at fertilization in Xenopus eggs is a propagating, Ca(2+)-dependent pH wave which closely follows the Ca2+ wave, together with the absence in the egg plasma membrane of pHi-regulating systems responsible for that pHi increase, suggest the existence of cortical or subcortical vesicles acidifying in the wake of the Ca2+ wave, thus producing the pH wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

Occurrence and activity of iron- and sulfur-oxidizing microorganisms in alkaline coal strip mine spoils

Spoils samples collected from a coal strip mine in southeastern Montana were examined for populations and activities of iron- and sulfur-oxidizing bacteria. Spoils examined were of three types: (a) acidic pyrite-rich waste coal, (b) oxidation halo material, and (c) alkaline material, which was the most widespread type. Bacterial numbers, sulfur oxidation, and¹⁴CO₂ uptake activity declined to low levels in the summer when spoils were dry. Even in wetter spring months pyritic spoils contained relatively low numbers of acidophilic iron- and sulfur-oxidizing bacteria, probably indicative of water stress since the same spoils incubated with excess water or dilute mineral salts showed considerably greater bacterial numbers and activity. Certain wells in coal and spoils aquifers contained substantial populations of iron-oxidizing acidophilic bacteria. However, these wells were always of alkaline or neutral pH, indicating that bacterial pyrite oxidation occurred where groundwaters contacted either replaced spoils or coal that contained pyrite or other metal sulfides. Bacterial activity may contribute to trace metal and sulfate leaching in the area.     

Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.     

Survival of two enterobacteria in feces buried in soil under field conditions.

Feces samples, inoculated with 10(6) Escherichia coli resistant to streptomycin and nalidixic acid and with 10(5) Salmonella typhimurium per g, were buried at five mountain field sites ranging from 2,005 to 2,730 m in elevation. Counts of each bacterium rose initially and then declined to 10(3) or 10(4) per g of feces in 8 weeks. The survival pattern was similar at all sites regardless of marked differences in elevation, soil, moisture, exposure, and vegetation. S. typhimurium numbers were consistently higher than E. coli numbers after week 3. The test encompassed most of the time that the area is snow-free and accessible for hiking. The results were judged to discredit the recommendation for shallow burial of feces and to indicate a potential health hazard under intensive use.