Nickel requirement for chemolithotrophic growth in hydrogen-oxidizing bacteria

In a comparative study the requirement of several strains of autotrophic hydrogen-oxidizing bacteria for nickel was examined. Autotrophic growth was studied both in liquid media, previously freed from trace metals; and on solidified media, using a plate diffusion assay. The latter assay was based on the observation that EDTA causes complete inhibition of autotrophic growth on agar medium as a result of nickel deficiency. Nickel was shown to be required as a trace element in five strains of Alcaligenes eutrophus, in two strains of Xanthobacter autotrophicus, in Pseudomonas flava, in Arthrobacter spec. 11X and in strain 12X. In these bacteria nickel was not replaceable by cobalt, copper, manganese or zinc ions. No significant nickel requirement was detected by these methods, however, for Paracoccus denitrificans and Nocardia opaca 1b.     

The action of alpha-ketoglutarate dehydrogenase on 4-chloronitrosobenzene: evidence for species-dependent differences in active site properties

The reaction of bovine (Bos taurus) and porcine (Sus scrufa) cardiac alpha-ketoglutarate dehydrogenase complex (alpha-KGD) with 4-chloronitrosobenzene (I) was shown to produce a hydroxamic acid (IV) and a product due to a Bamberger rearrangement as previously shown for Escherichia coli alpha-KGD. The conversion of I into an active site-bound electrophile was general among the three alpha-KGD enzymes tested, but quantitative differences in products and kinetics were shown. The reaction of I was specific for the resolved alpha-ketoglutarate decarboxylase subunit.     

On the sulfur-source requirement for growth of Thiobacillus intermedius

Thiobacillus intermedius grows in glucose-mineral salts medium supplemented with any or all of seven reduced organic sulfur compounds but does not grow in the absence of the supplement(s). This growth represents a physiological capability of T. intermedius and does not result from contaminated or mutant cultures. T. intermedius is not capable of assimilating sulfate; exogenous ³⁵SO₄ ^-is not incorporated by T. intermedius growing autotrophically or heterotrophically. The demonstrated need for a reduced sulfur source explains the previously reported inability of T. intermedius to grow in glucose-mineral salts medium.     

Platelets induce reactive oxygen species-dependent growth of human skin fibroblasts

A growing amount of evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, regulate intracellular signalling and have a role in cell proliferation. In the present study, we show that platelets increase the mitogenic rate in human fibroblasts and that this effect was inhibited by the intracellular antioxidant N-acetyl-L-cysteine (NAC) and the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). The mitogenic effects of platelets were mimicked by the platelet factors platelet-derived growth factor BB-isoform (PDGF-BB), transforming growth factor beta1 (TGF-beta1) and sphingosine-1-phosphate (S1P). The sphingosine kinase inhibitor DL-threo-dihydrosphingosine (DL-dihydro) abrogated the platelet-induced growth, while antibodies directed against PDGF or TGF-beta had modest effects. Exposure of fibroblasts to platelets, PDGF-BB, TGF-beta1 or S1P caused an extensive intracellular ROS production, measured as changes in dichlorofluorescein fluorescence. This ROS production was totally inhibited by NAC, pyrrolidinethiocarbamate (PDTC), DPI and apocynin. In conclusion, the results presented are indicative of a crucial role of ROS in the platelet-mediated regulation of fibroblast proliferation.     

Minimal nutrient requirement for growth ofMycobacterium chlorophenolicum

Summary The nutrient demand of the pentachlorophenol-degrading bacteriumMycobacterium chlorophenolicum was characterized in shake flask experiments. A minimal medium containing sorbitol as sole carbon and energy source and thiamine as the only organic growth factor was developed that allows satisfactory growth of the strain.     

Kinase requirement for retinal growth cone motility

Since cytoplasmic Ca²⁺ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantifed over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1–1 μM) and chelerythrin (up to 50 μM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 μM), trifluoperazine (100 μM), and CGS9343B (50 μM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 μM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 μM), produced no effects but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3–5 μM) and ML-9 (5–10 μM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 μM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts. 1994 John Wiley & Sons, Inc.     

Glutamine requirement for aerial mycelium growth in Neurospora crassa

Five amino acids are accumulated during vegetative growth of Neurospora crassa, particularly.during the prestationary growth phase. Alanine, glutamine, glutamate, arginine and ornithine.comprised over 80% of the total amino acid pool in the mycelium. Amino acid pools of different amino acid auxotrophs were followed during the partial transformation of a mycelial mat into an aerial mycelium. The mycelial mat under starvation and in direct contact with air rapidly formed aerial mycelium, which produced thereafter a burst of conidia. During this process,glutamine and alanine in the mycelial mat were consumed more rapidly than other amino acids;in the growing aerial mycelium, glutamate and glutamine were particularly accumulated. Of the amino acids that were initially accumulated in the mycelial mat, only a high glutamine pool was required for aerial mycelium growth induced by starvation. This requirement for glutamine could not be satisfied by a mixture of the amino compounds that are synthesized via glutamine amidotransferase reactions. It is proposed that glutamine serves as a nitrogen carrier from the mycelial mat to the growing aerial mycelium.     

Oxygen and reactive oxygen species-dependent regulation of plant growth and development

Oxygen and reactive oxygen species (ROS) have been co-opted during evolution into the regulation of plant growth, development, and differentiation. ROS and oxidative signals arising from metabolism or phytohormone-mediated processes control almost every aspect of plant development from seed and bud dormancy, liberation of meristematic cells from the quiescent state, root and shoot growth, and architecture, to flowering and seed production. Moreover, the phytochrome and phytohormone-dependent transmissions of ROS waves are central to the systemic whole plant signaling pathways that integrate root and shoot growth. The sensing of oxygen availability through the PROTEOLYSIS 6 (PRT6) N-degron pathway functions alongside ROS production and signaling but how these pathways interact in developing organs remains poorly understood. Considerable progress has been made in our understanding of the nature of hydrogen peroxide sensors and the role of thiol-dependent signaling networks in the transmission of ROS signals. Reduction/oxidation (redox) changes in the glutathione (GSH) pool, glutaredoxins (GRXs), and thioredoxins (TRXs) are important in the control of growth mediated by phytohormone pathways. Although, it is clear that the redox states of proteins involved in plant growth and development are controlled by the NAD(P)H thioredoxin reductase (NTR)/TRX and reduced GSH/GRX systems of the cytosol, chloroplasts, mitochondria, and nucleus, we have only scratched the surface of this multilayered control and how redox-regulated processes interact with other cell signaling systems.

Oxygen and reactive oxygen species regulate plant growth, development, and differentiation through multiple interlinked signaling pathways.

Advances

• Developmentally regulated hypoxia and reactive oxygen species (ROS) production are key features of the stem cell niches, providing information about stem cell position, the environment, and metabolic state.
• Protein cysteine oxidation is central to oxygen and ROS signaling. However, S-nitrosylation, S-glutathionylation, S-sulfhydration, and S-sulfenylation modifications can occur on the same cysteine. The influence of each modification on stability, localization, and function remains unknown.
• Numerous intersecting ROS signaling pathways are probable and likely depend on the site of ROS production and the nature of the oxidized receptor protein. ROS sensors such as the hydrogen peroxide (H₂O₂)-INDUCED Ca²⁺ INCREASES 1 (HPCA1) leucine rich receptor kinase translate redox signals into protein modifications to regulate signaling cascades. H₂O₂ perception/transduction is dependent on thiol-dependent mechanisms policed by the ferredoxin/thioredoxin (TRX), NAD(P)H TRX reductase C (NTRC), reduced glutathione (GSH), and glutaredoxin (GRX) systems.
• ROS waves transmit redox signals from cell to cell in the apoplast, and probably through plasmodesmata. Long-distance transport of H₂O₂ and other ROS, therefore, appears to be unnecessary. Similarly, contact sites between organelles allow ROS transfer.
• Convergence points for oxygen and ROS signaling occur on proteins such as ROH OF PLANT 2 (ROP2) GTPase,RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), and TRX-h to regulate meristematic activity via TARGET OF RAPAMYCIN (TOR) kinase activity.

     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts

U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF(35) subunit contacts the 3'-splice site AG. Here we show that U2AF35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.     

The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abl's effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia.     

Identification of the amino acid residues involved in the species-dependent differences in the pyridoxine transport function of SLC19A3

Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln⁸⁶, Gly⁸⁷, Ile⁹¹, Thr⁹³, Trp⁹⁴, Ser¹⁶⁸, and Asn¹⁷³ are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.     

Transient growth requirement in Bacillus subtilis following the cessation of exponential growth

During an investigation of the parameters controlling mutations in Bacillus subtilis we observed that this bacterium exhibits a transient growth requirement for two nonessential amino acids (glutamic acid and isoleucine) during a type of postexponential growth on a minimal medium.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.