Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.     

Metronidazole and the isolation of temperature-sensitive photosynthetic mutants in cyanobacteria

A procedure has been developed for use of metronidazole (2-methyl-5-nitroimidazole-1-ethanol) as an enrichment agent during the isolation of temperature-sensitive, photosynthetic mutants in the cyanobacteriumSynechococcus cedrorum. The protocol includes incubation with this drug following mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Incubation of photosynthetically activeS. cedrorum cells with 1 mM metronidazole causes a light-dependent reduction of cell viability. Maximum reduction in cell viability occurred following 6 h of incubation. Cessation of electron transport reduced the impact of the drug by five orders of magnitude. Yet during the time of incubation, metronidazole did not influence the electron transport capacities of theS. cedrorum cells, suggesting that the thylakoid membrane was not the target of the toxic effects of this drug. In addition, this drug was found to be an effective electron acceptor to photosystem I although high concentrations were required to observe maximum rates of electron transfer. Metronidazole interacted in a noncompetitive manner with methyl viologen, which suggested that those two acceptors to photosystem I have unique reduction sites on theS. cedrorum thylakoid membrane. The temperature-sensitive strains that were isolated using the procedure presented here were assessed for photosynthetic electron transport and chlorophyll fluorescence (induction kinetics and low-temperature emission spectra) characteristics. Approximately one-half of the temperature-sensitive mutants isolated possessed abnormal photosynthetic properties when shifted to the restrictive temperature (40°C). A total of 31 strains have been characterized and initially classified, showing abnormalities throughout the photosynthetic electron-transport chain.     

Isolated water in desiccated microorganisms

The presence of isolated mobile water in dehydrated eukaryotic microorganisms established earlier by NMR has been confirmed by direct chemical registration of this water in the yeast Cryptococcus albidus. This water constitutes several per cent of the dry biomass weight. Apparently, its preservation should be attributed to changes in the permeability of intracellular membranes upon dehydration. The water is released by the cells when they are heated to 150--200 degrees C and the cellular structures containing water are destroyed.     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

Development and differentiation of mouse blastocysts in serum-free medium

Numerous studies have shown that the in vitro development and differentiation of mouse blastocysts require serum, but the number and nature of serum factors involved remains unclear. In this article, we describe a culture medium, EM-2, containing as a source of protein only commercially purified bovine serum albumin (BSA) and fetuin. This medium supports hatching, attachment and outgrowth of mouse blastocysts. Although attachment and outgrowth are delayed in EM-2 medium 12–15 and 5–8 h, respectively, these events occur at frequencies comparable to those observed in serum-containing media. Trophoblast cells are capable of differentiating in this medium: they synthesize Δ⁵,3β-hydroxysteroid dehydrogenase and their nuclei become polyploid. The inner cell mass also appears to differentiate to some extent in EM-2 medium as evidenced by the appearance of cells with characteristics of parietal endoderm. The fetuin factor is necessary at least for trophoblast outgrowth and the albumin factor is required for the survival and/or growth of the inner cell mass. It is, however, not evident from these studies whether the serum fractions used are actually involved in the induction of differentiation, or whether the early differentiative steps in the mouse blastocyst are preprogrammed and require for expression only a normal cellular metabolic rate.     

Steroid-receptor quantitation and characterization by electrophoresis in highly cross-linked polyacrylamide gels.

Conditions for discontinuous polyacrylamide gel electrophoresis have been defined in which progesterone receptors of chick oviduct cytosol and a variety of steroid-binding proteins from other sources are stable and amenable to quantitative analysis. The essential modifications from standard procedures include the use of (1) separation gels in which the cross-linking agent/acrylamide monomer = 15:85, (2) glycerol (10% v/v) in all phases of the Trisglycine-HCl buffer system (pH 10.2 in the separation phase during electrophoresis at 0 degrees), and (3) a layer of a charged reducing agent, thioglycolate, beneath the sample layer. Electrophoresis of untreated oviduct cytosol labeled with [3H]progesterone +/- competing steroids revealed a heterodisperse slow peak and a sharp fast peak. Both peaks displayed the steroid-binding specificity and saturability that are characteristic of intracellular receptors. Recovery of steroid from both the slow and fast components increased linearly with sample load up to 60 mul of cytosol (1.2 mg of protein)/gel (6 mm diameter). The specific progesterone binding detected by this technique was comparable to that detected by charcoal-dextran treatment or ion exchange filtration. Relative electrophoretic mobilities (Rf) of globular protein standards and steroid-protein complexes in cytosol and chick serum were measured in separation gels with total gel concentrations (T) systematically varied from 5 to 15% (w/v). Data were processed by computer programs to obtain weighted linear regressions of log Rf on T (Ferguson plots) and the joint 95% confidence limits of the slopes (-KR) and intercepts of these plots. Molecular radii (R) of the binding components and apparent molecular weights (M) were calculated from the linear correlation of R with KR 1/2 for the standards. The value of M is approximately 158,000 obtained for the cytosol fast component was independent of the length of the separation gel, the presence of a stacking gel or prior exposure of the cytosol to KCl. It was higher than expected from the sedimentation coefficient of 4.2 S in the same pH 10.2 buffer. Electrophoresis in 170-mm separation gels without stacking gels revealed that KCl extracts of protamine-precipitated cytosol contain a different receptor form, of lower net negative charge than the cytosol fast form. The results demonstrate the utility of electrophoresis in highly cross-linked gels of several concentrations to discriminate between various receptor forms and steroid-binding components of serum. This method may lead to overestimates of M for highly asymmetric receptor forms.     

A modified walking rhythm employed during righting behavior in the cockroachGromphadorhina portentosa

Summary Electromyograms were recorded from leg muscles of the cockroachGromphadorhina during walking and righting under free-ranging and tethered conditions. Two muscles which are essentially synergistic during walking become antagonistic during righting (Fig. 3, 4). This explains in part the difference in the direction of the leg stroke in the two behaviors (Fig. 2). Other properties of the muscle activity are very similar during the two rhythms: the same motoneurons appear to be active (Fig. 5, 6); cycle frequencies are the same; the burst length of one motoneuron studied varies with burst frequency in a generally similar manner in both behaviors (Fig. 7); inter-leg coordination is the same (Fig. 9); and transganglionic coupling characteristic of walking can occur while a leg on one side is engaged in walking, and its contralateral homologue is engaged in righting (Fig. 10). Although other properties of the leg rhythms are different in walking and righting, these differences appear to result from dissimilarities in sensory feedback. It is concluded that although the two leg rhythms are superficially quite different, the underlying central neuronal rhythms are very similar, and possibly result from activity in the same central oscillatory cell or circuit.We thank Carol Smith for technical assistance. This work was supported by NIH grant #NS09083-05. Computation was done at the New York State Veterinary College Computer Facility which is supported by NIH grant RR 326.