Effects of bud removal in Scots pine (Pinus silvestris) seedlings

One- and two-“year”-old seedlings of Pinus silvestris L., from which the buds had been removed, were studied for five weeks during the second and third growth period, respectively. Intact seedlings were used as controls. The seedlings were cultivated under controlled conditions in a climate chamber. The growth of the seedlings was determined and the one-“year”-old needles assayed for changes in net photosynthesis and ribulose bisphosphate carboxylase activity and in the levels of protein, Kjeldahl nitrogen, chlorophyll and starch. In the control the carboxylase activity and the content of protein, Kjeldahl nitrogen and starch in the needles increased in the beginning of the “summer” and decreased during the shoot growth period. The starch content was higher after bud removal (decapitation), since the carbohydrate could not be utilized for the growth of the new shoot. Decapitation did not affect the growth rate of the roots. The content of Kjeldahl nitrogen and total and soluble protein in the needles was higher in the decapitated seedlings during the period of shoot elongation in the control. Total nitrogen, but not protein, reached high levels, indicating accumulation of non-protein compounds. The general course of the chlorophyll pattern was not affected. Higher ribulose bisphosphate carboxylase activity than in the control was observed in the later part of the experimental periods. The higher levels of protein and nitrogen as well as of carboxylase activity after decapitation support the interpretation that soluble protein, including the carboxylase, and possibly other nitrogen compounds in the older needles are used for growth of the shoot. The loss of protein and nitrogen and of carboxylase activity in the control did not seem to be due to mineral deficiency in the substrate. Despite higher levels of carboxylase activity and similar chlorophyll concentrations, light-saturated net photosynthesis was lower after decapitation. The ratio between photosynthesis and photorespiration was not affected.     

Metabolism of fatty acids and their incorporation into phospholipids of the mitochondria and endoplasmic reticulum in isolated hepatocytes determined by isolation of fluorescence derivatives

Isolated hepatocytes were incubated in the presence of [14C]palmitic, [14C]linoleic or [14C]linolenic acid and the time-courses of incorporation of radioactivity into phosphatidylcholine and phosphatidylethanolamine of microsomes and mitochondria were followed. For this purpose a procedure was developed for HPLC separation of 9-diazomethylanthracene (ADAM) derivatives of fatty acids. When [14C]palmitic acid was used, the major product of elongation and desaturation was octadecadienoic acid, which accounted for 35-65% of the total radioactivity. Labeled palmitoleic, stearic and oleic acids could also be isolated. In fatty acids which do not participate to any large extent in deacylation-reacylation reactions, the pattern of incorporation was characteristic: a high rate of incorporation into microsomal and a low rate of incorporation into mitochondrial phospholipids during the first 40 min, followed by a decrease in the former and an increase in mitochondrial labeling. This pattern is consistent with the fact that de novo synthesis of these two phospholipids occurs in the endoplasmic reticulum in vivo. When cells were incubated in the presence of [14C]linoleic acid, 70-90% of the radioactivity recovered in phospholipids was in this same form, whereas the remaining label was mainly in arachidonic acid and, to some extent, in eicosatrienoic acid. When hepatocytes were incubated in the presence of [14C]linolenic acid, 70-85% of the radioactivity in isolated phospholipids was associated with linolenic acid. As much as 20% of the label was recovered in docosahexanoic acid and 5-10% in arachidonic acid. In the case of the two latter labeled substrates the exchange reactions seem to dominate over de novo synthesis. For phospholipids synthesized de novo the transfer from the endoplasmic reticulum to mitochondria requires about 3 h.     

Electron microscopic demonstration of acid phosphatase activity in the developing and mature heterophils of the chicken

Summary As shown by electron microscopic histochemistry using a modified Gomori lead salt technique, acid phosphatase is present in large dense granules and the Golgi apparatus —but not the light granules—in both immature and mature heterophils in the chicken. The large dense granules appear to form by budding from the Golgi cisternae while the light granules appear to be unassociated with the Golgi apparatus. The findings indicate that the large, dense granules are the lysosomes of the heterophils in the chicken.     

Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8

The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented. The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides. The sequence is homologous to c-type cytochromes, especially in the heme-binding region.     

Vertebrate head development: Segmentation,novelties, and homology

Vertebrate head development is a classical topic lately invigorated by methodological as well as conceptual advances. In contrast to the classical segmentalist views going back to idealistic morphology, the head is now seen not as simply an extension of the trunk, but as a structure patterned by different mechanisms and tissues. Whereas the trunk paraxial mesoderm imposes its segmental pattern on adjacent tissues such as the neural crest derivatives, in the head the neural crest cells carry pattern information needed for proper morphogenesis of mesodermal derivatives, such as the cranial muscles. Neural crest cells make connective tissue components which attach the muscle fiber to the skeletal elements. These crest cells take their origin from the same visceral arch as the muscle cells, even when the skeletal elements to which the muscle attaches are from another arch. The neural crest itself receives important patterning influences from the pharyngeal endoderm. The origin of jaws can be seen as an exaptation in which a heterotopic shift of the expression domains of regulatory genes was a necessary step that enabled this key innovation. The jaws are patterned by Dlx genes expressed in a nested pattern along the proximo-distal axis, analogous to the anterior–posterior specification governed by Hox genes. Knocking out Dlx 5 and 6 transforms the lower jaw homeotically into an upper jaw. New data indicate that both upper and lower jaw cartilages are derived from one, common anlage traditionally labelled the “mandibular” condensation, and that the “maxillary” condensation gives rise to other structures such as the trabecula. We propose that the main contribution from evolutionary developmental biology to solving homology questions lies in deepening our biological understanding of characters and character states.     

Competitive inhibitors of Mycobacterium tuberculosis ribose-5-phosphate isomerase B reveal new information about the reaction mechanism

Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (K(m) 57 microm) and 4-phospho-d-erythronate (K(i) 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.     

Opening of mitochondrial KATP channels enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation, and respiration

Previously, we have shown that the pharmacological opening of the mitochondrial ATP-sensitive K channels with diazoxide (DZX) enhances the cardioprotection afforded by magnesium-supplemented potassium (K/Mg) cardioplegia. To determine the mechanisms involved in the cardioprotection afforded by K/Mg + DZX cardioplegia, rabbit hearts (n=24) were subjected to isolated Langendorff perfusion. Control hearts were perfused for 75 min. Global ischemia (GI) hearts were subjected to 30 min of equilibrium, 30 min of GI, and 15 min of reperfusion. K/Mg and K/Mg + DZX cardioplegia hearts received either K/Mg or K/Mg + DZX for 5 min before GI and reperfusion. Tissue was harvested for mitochondrial isolation and transmission electron microscopy (TEM). Mitochondrial structure, area, matrix volume, free calcium, and oxygen consumption were determined. TEM demonstrated that GI mitochondria were damaged and that K/Mg and K/Mg + DZX preserved mitochondrial structure. TEM and light scattering demonstrated separately that mitochondrial matrix and cristae area and matrix volume were significantly increased after GI and reperfusion with GI > K/Mg + DZX > K/Mg hearts (P <0.05 vs. control). Mitochondrial free calcium was significantly increased in GI and K/Mg hearts. K/Mg + DZX significantly decreased mitochondrial free calcium accumulation (P <0.05 vs. GI and K/Mg). State 3 oxygen consumption and respiratory control index in malate (complex I substrate)- and succinate (complex II substrate)-energized mitochondria were significantly decreased (P <0.05 vs. control) in the GI and K/Mg + DZX groups. These data indicate that the enhanced cardioprotection afforded by K/Mg + DZX cardioplegia occurs through the preservation of mitochondrial structure and the significant decrease in mitochondrial free calcium accumulation and mitochondrial state 3 oxygen consumption.     

Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 µM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states. iron transport; HepG2 cells     

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope

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