Synchronous mitochondrial division in Neurospora crassa.

Samples of mycelium of Neurospora crassa of known age were harvested from agar plates and examined with the electron microscope. The relative volume of the mitochondria was determined for mycelium of different ages. The volume measurements indicated that the mitochondria were dividing synchronously in fronts 6, 13, and 22(1/2) hr behind the growing hyphal tips. The sequence of mitochondrial division is hypothesized to include mitochondrial cupping followed by division which results in closely associated daughter mitochondria. On the basis of percentages of mitochondrial cupping and association, mitochondrial division was postulated to be occurring at 6, 14, and 26 hr. Close agreement between the mycelial mass doubling time and the calculated mitochondrial mass doubling time indicates that synchronous mitochondrial division is sufficient to maintain growth. The possibility that mitochondrial division is due to intercellular regulation of a mitochondrial genetic system is advanced.

Separation of mitochondrial membranes of Neurospora crassa. II. Submitochondrial localization of the isoleucine-valine biosynthetic pathway.

Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.

Phagocytosis and growth inhibition of Cryptococcus neoformans by human alveolar macrophages: effects of HIV-1 infection.

OBJECTIVE: The purpose of this study was to investigate the in vitro processing of Cryptococcus neoformans by human alveolar macrophages from HIV-seropositive individuals compared with HIV-seronegative individuals. DESIGN AND METHODS: Bronchalveolar lavage (BAL) was performed on smoking and nonsmoking HIV-seropositive and seronegative volunteers. Lavage cells from the four groups were challenged in vitro with C. neoformans and assessed for fungal binding, phagocytosis, and growth inhibition. RESULTS: The results indicated that BAL cells from the smoking HIV-infected group had increased fungistatic activity compared with HIV-seronegative smokers (mean percentage growth inhibition +/- SD, 77.5 +/- 14.2 versus 59.1 +/- 16.6%; P = 0.015). However, late-staged HIV-infected patients (Centers for Disease Control and Prevention class C3) were found to have decreased fungistasis compared with early stage A patients (63.8 +/- 11.1 versus 83.0 +/- 2.2%; P < 0.05). BAL cells recovered from HIV- seronegative smoking volunteers demonstrated reduced fungistatic activity when compared with BAL cells from HIV- seronegative nonsmokers (56.8 +/- 8.8 versus 83.0 +/- 2.2%; P < 0.001). Smoking also induced a decrease in internalization of C. neoformans by alveolar macrophages as assessed by confocal laser microscopy in both HIV-seronegative and HIV-infected groups. CONCLUSION: We conclude that BAL cells from early stage HIV-1-infected individuals do not have an intrinsic defect in fungistasis of C. neoformans. In fact, it appears that BAL cells from HIV-seropositive people are activated for fungistasis in early HIV infection, although fungistatic activity declines as the disease progresses. Incidentally noted was the finding that smoking decreases the internalization of C. neoformans in both HIV-infected and HIV-seronegative individuals, suggesting the possibility that smoking might enhance the susceptibility to cryptococcosis.

Waldenstrom macroglobulinemia presenting as a pleural effusion.

We describe a patient with Waldenstrom macroglobulinemia who presented with a lymphocytic pleural effusion. Pleural involvement in Waldenstrom macroglobulinemia is very rare. In addition, to our knowledge, there are no reports in the literature in which a primary diagnosis was made on the basis of pleural fluid analysis. The presence of small lymphocytes in serous cavity fluid can pose great difficulty in the differentiation between a low-grade lymphoproliferative disorder and reactive lymphocytosis. The diagnosis of malignancy in this case was established on the basis of morphologic testing, flow cytometry, and the detection of B-cell immunoglobulin gene rearrangement.

Studies on the regulation of the branched chain amino acyl-tRNA synthetases of the fungusNeurospora crassa.

The specific activities of the branched chain amino acyl-tRNA synthetases from the cytosolic and mitochondrial fractions ofN. crassa were low in dormant conidia and increased during germination, reaching a maximum 8 h after inoculation. This stage of development is characterised by high rates of many other cellular activities.The increases in activity of synthetases of both cytosol and mitochondria are inhibited by cycloheximide indicating that they are synthesized on cytoplasmic ribosomes. The mitochondrial synthetases show a stimulation of their specific activity when mitochondrial RNA and protein synthesis are inhibited by either ethidium bromide or chloramphenicol suggesting that a mitochondrial translation product regulates the synthesis of the mitochondrial synthetases.The activities of amino acyl-tRNA synthetases are dependent on energy production. When respiration is uncoupled from oxidative phosphorylation, synthetase specific activities decrease although the activities of other mitochondrial enzymes like NADH-dehydrogenase increase. This phenomenon suggests that more than one mechanism regulates the synthesis of mitochondrial proteins which are formed on cytoplasmic ribosomes.The synthesis of branched chain amino acyl-tRNA synthetases ofNeurospora is neither repressed by their cognate amino acids, nor is there inhibition by the precursors of these amino acids, as has been observed in other amino acyl-tRNA synthetases of various organism includingNeurospora.

Two forms of a mitochondrial endonuclease in Neurospora crassa.

Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods.

Thymidine-kinase activity of cultured cells from individuals with inherited galactokinase deficiency.

Cells of a person homozygous for galactokinase deficiency and of her heterozygous parents were found to be deficient in the enzyme thymidine kinase. The decrease in thymidine-kinase activity may be the result of a qualitative alteration in the enzyme molecule. This is reflected in the apparent alteration in the sensitivity of the enzyme to trifluorothymidine. It is suggested that this relationship between the galactokinase and thymidine kinase is not fortuitous but a reflection of their interdependence as found previously in the Chinese hamster.

Acetohydroxy acid synthetase with a pH optimum of 7.5 from Neurospora crassa mitochondria: characterization and partial purification.

An acetohydroxy acid synthetase (AAS) has been found associated with the mitochondrial fraction of wild-type Neurospora crassa. It has a pH optimum of 7.5 and is presumed to be homologous to the pH 8.0 AAS that synthesizes the valine and isoleucine precursors in bacteria and yeast. The enzyme was characterized and purified 30- to 60-fold. The AAS activity of intact mitochondria requires thiamine pyrophosphate (TPP), Mn(2+) or Mg(2+), and flavine adenine dinucleotide (FAD), and is sensitive to end product inhibition by l-valine. This inhibition is pH-dependent and noncompetitive with respect to pyruvate. Activity is slightly repressed during exponential growth in the presence of valine, isoleucine, and leucine. Extraction of the AAS from the mitochondria has a profound influence on the following properties: pH optimum, sensitivity to l-valine, response to FAD, binding of TPP, apparent K(m), and stability at 0 to 4 C. The catalytic properties of the partially purified enzyme are described. Two forms of the partially purified AAS can be isolated from preparative Sephadex G-200 chromatographic columns. Both forms are electrophoretically and antigenically similar but one form has an estimated molecular weight of 110,000 to 120,000 whereas the predominant form is a much larger and more buoyant molecule.

A coordinate relationship between the GALK and the TK1 genes of the Chinese hamster.

Chinese hamster cells in culture were treated with various concentrations of thymidine, 5-bromodeoxyuridine, trifluorothymidine, and 2-deoxy-D-galactose. Selection was made for deficiencies in the activities of galactokinase and thymidine kinase. Selection in the presence of thymidine, 5-bromodeoxyuridine, and trifluorothymidine was expected to produce clones deficient in thymidine kinase only, whereas those deficient in galactokinase were expected to be selected in the presence of 2-deoxy-D-galactose. However, it was found that clones growing in the presence of these inhibitors were frequently deficient in both enzymes. Or if a clone was deficient in only one, the deficiency frequently was not expected according to the selection procedure. This indicates some sort of coordinate relationship between the two gene loci, GALK and TK1, which specify galactokinase and thymidine kinase, respectively. GALK and TK1 are linked in all primates and rodents in which linkage determinations have been made. It is therefore probable that this linkage has been conserved for a long period of time. It is suggested that the apparent relationship between the two genes shown by the data presented here, as well as by others, supports the conclusion that linkage has been conserved by natural selection and is therefore not fortuitous.