Discovery,localization, and sequence characterization of molecular markers for the crown rust resistance genes <Emphasis Type="Italic">Pc38, Pc39</Emphasis>, and <Emphasis Type="Italic">Pc48</Emphasis> in cultivated oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Molecular markers for the crown rust resistance genes Pc38, Pc39, and Pc48 in cultivated oat (Avena sativa L.) were identified using near-isogenic lines and bulked segregant analysis. Six markers for Pc48, the closest being 6 cM away, were found in a Pendek-39 × Pendek-48 (Pendek3948) population, but none was found in a Pendek-48 × Pendek-38 (Pendek4838) population. Three markers for Pc39 were found in the Pendek3948 population, one of which cosegregated with the gene. This same marker was found to be 6 cM away from the gene in an OT328 × Dumont (OT328Du) population. Nine markers for Pc38 were found in the Pendek4838 population, eight of which are within 2 cM of the gene. One other marker for Pc38 was found in the OT328Du population; however, comparative mapping suggests that the Pc38 region in OT328Du is in a different location than that in Pendek4838. A number of markers unlinked to the genes under study formed linkage groups in both the Pendek3948 and Pendek4838 populations. Four of these show homology or homoeology to each other and to the Pc39 region in Pendek3948. Two RFLP clones closely linked to Pc38 code for a putative leucine-rich repeat transmembrane protein kinase and a cre3 resistance gene analogue. This study provides information to support molecular breeding in oat, and contributes to ongoing research into genomic regions associated with fungal pathogen resistance.     

Ultrastructural localisation of TGF-beta exposure in dentine by chemical treatment

Transforming growth factor- (TGF-) sequestered in dentine matrix has an important role in dental tissue repair after injury and its exposure at sites of injury may stimulate tertiary dentinogenesis. This study aimed to investigate the expression of TGF- isoforms in mature human dentine matrix and the ability of chemical treatments to expose TGF- on the cut surface of dentine using gold immunolabelling and subsequent scanning electron microscopy examination. TGF-1 was the only isoform that could be detected in human dentine and the nature of the chemical treatment of the tissue influenced its detection. EDTA treatment provided good exposure of TGF-1 on the dentine surface, whilst citric acid and sodium hypochlorite treatments revealed lesser amounts of this isoform. Only minimal staining for TGF-1 was observed in samples treated with phosphate-buffered saline. TGF-2 and -3 could not be detected in the specimens with any of the treatments. This study suggests that TGF-1 is the only TGF- isoform expressed by human odontoblasts to be sequestered in dentine implying that differences in isoform–extracellular matrix interactions may exist. Information on chemical treatment of tissue specimens for immunostaining may provide a useful basis for selection of tissue preparation techniques for clinical restorative treatment procedures to facilitate TGF- mediated reparative processes at sites of dental injury.     

Über den Einfluß des Salzgehaltes auf die photosynthetische Leistung verschiedener Standortformen vonDelesseria sanguinea undFucus serratus

Zusammenfassung 1. Es wurde untersucht, welchen Einfluß kurzfristige und langfristige Salzgehaltsveränderungen auf verschiedene Standortformen der RotalgeDelesseria sanguinea und der BraunalgeFucus serratus haben. Als Kriterium des Lebenszustandes wurde die photosynthetische Leistung gewählt. Die Algen wurden folgenden Salzgehaltskonzentrationen ausgesetzt: 0, 5, 10, 15, 20, 30, 40, 50 S.2. Die Versuche ergaben, daß kurzfristige Konzentrationsveränderungen (30 min) — sowohl Erniedrigung als auch Erhöhung des Salzgehaltes — die photosynthetische Leistung stimulieren. Ein langfristiger Aufenthalt (24 Std) unter den veränderten Bedingungen bewirkt, sofern diese innerhalb der Toleranzgrenzen der Algen liegen, einen Ausgleich der anfänglichen Stimulation. Außerhalb der Toleranzgrenzen liegende Konzentrationen rufen nach der Stimulation eine Leistungsdepression hervor. Bei Rückübertragung in den Ausgangssalzgehalt sind die Depressionen teilweise reversibel.3. Im hypotonischen Milieu verhalten sich die Delesserien der verschiedenen Standorte (Helgoland, Kattegat, Kieler Bucht) gleich: in 5 S treten starke Depressionen auf. Nordsee-Delesserien sind im hypertonischen Milieu weniger empfindlich, sie zeigen noch bei 50 S eine gesteigerte photosynthetische Leistung. In diesem Bereich sind die Ostseeformen schon schwer geschädigt. Am empfindlichsten gegenüber allen Konzentrationsänderungen ist die BrackwasserformDelesseria sanguinea formalanceolata aus der Kieler Bucht.4.Fucus serratus aus dem Litoral von Helgoland zeichnet sich im Gegensatz zu der submers lebenden Form der Ostsee, die sich ähnlich wieDelesseria verhält, in allen untersuchten Konzentrationsbereichen durch eine unveränderte photosynthetische Leistung aus. Die beiden Standortformen vonFucus entsprechen gemäß der Einteilung vonMontfort (1931) dem resistenten Typ und dem Stimulations-Depressionstyp.

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On the influence of salinity on photosynthetic performance of various ecotypes ofDelesseria sanguinea andFucus serratus

The phaeophyceanF. serratus and the rhodophyceanD. sanguinea came from the North Sea (30 S) and the Baltic Sea (15 S). The activity of photosynthesis was taken as a criterion of algae vitality. Experiments were made in salinity concentrations of 0, 5, 10, 15, 20, 30, 40 and 50 S. Thirty-minute exposures to sub- or supranormal salinities stimulate photosynthesis. Within their physiological salinity ranges the algae assume normal photosynthetic rates within 24 hours. Extreme salinities cause a reduction in photosynthetic activity; this reduction mostly disappears, however, after re-transfer into normal salinity conditions. At 5 S all test individuals ofDelesseria from different locations exhibit a reduction of photosynthetic rates. At 50 SDelesseria from the North Sea still show increased activity, whileDelesseria from the Baltic are already severely damaged. The brackish-water formD. sanguinea (formalanceolata) is most sensitive to salinity variations. The photosynthetic activity ofF. serratus from Helgoland does not vary in all salinities employed. The range of test salinities corresponds to that of the habitat in the littoral zone, where high salinities occur during air exposure, and low salinities, during rainfall. By contrast, inF. serratus from the Baltic Sea occurring only in the sublittoral zone, photosynthetic rates are similarly affected by salinity as inDelesseria.

     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-γ

We investigated the influence of the combined use of sizofiran, a-1,3-glucan and a recombinant interferon- (rIFN-) upon biological activities of peritoneal macrophages (M). The number of peritoneal M and the production of cytokines (interleukin-1, interferon- and tumor necrosis factor) was increased by the combined treatment. Fully activated peritoneal M based on the increased number of elongated pseudopods were observed by electromicroscope. Sizofiran seems to assure a sufficient supply of M to kill tumor cells in the peritoneal cavity and co-administered rIFN- seems to directly stimulate the accumulated M in addition to its direct cytotoxicity against tumor cells. This combination therapy may be a step to the prevention of the recurrence of gynecological malignancies including ovarian cancer, after a negative second-look laparotomy.Abbreviations rIFN- recombinant interferon- - IL-1 interleukin-1 - TNF tumor necrosis factor - SLL second look laparotomy     

Ultrastructural studies on the adrenal medulla of golden hamster: Origin and fate of secretory granules

Summary Several different fixatives were used in order to obtain the best preservation of fine structure in the chromaffin cells of hamster adrenal medulla. The best fixative for both immersion-fixation and perfusion-fixation contained glutaraldehyde (2.5%) and formaldehyde (4%). After fixation by immersion of the gland, both dark and light cells are found, but glands fixed by perfusion contain a homogeneous population of light cells, which were very well preserved.The plasma membrane along the free surface of chromaffin cells showed a large number of omega-shaped invaginations that usually contained a dense core or fibre-like material; the extracellular dense cores were very similar to those of intact secretory granules. Rarely, the extracellular dense cores were very large and resembled the contents of a secondary lysosome. Several coated pits were found on the inner surface of each omega-shaped invagination.A prominent Golgi zone, containing many coated vesicles, is typical of these chromaffin cells. The coated vesicles are of two kinds, one with and one without electron-dense contents. Coated vesicles were frequently found in close contact with, or fused with, pro-secretory granules.Both authors are Wellcome Research Fellows. This work is supported by a grant from the Medical Research Council. We appreciate the technical assistance of Mr. P. T. Edwards.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Ultrastructure of carotenoid deprivation in photoreceptors of Manduca sexta: myeloid bodies and intracellular microvilli

Summary The ultrastructural effects of carotenoid (vitamin A) deprivation were studied in the adult photoreceptors of the tobacco hornworm moth Manduca sexta. Moths were reared on a deprived diet, which lacked the carotenoid sources of the photopigment chromophore, 3-hydroxy retinal, or on a control, fortified diet, containing ample carotenoid. The latter supported normal levels of visual function, whereas visual pigment and sensitivity were reduced by at least 3 log units in moths reared on the deprived diet. Myeloid bodies, massed cisternae of hypertrophied smooth endomembrane, filled the cytoplasm in the receptors of deprived animals. The myeloid bodies assumed various configurations that included lamellate stacks of parallel cisternae, and tubular networks in a paracrystalline form. Freeze-fracture preparations of myeloid membranes revealed a high density of P-face particles. Vacuoles containing microvilli similar to those of the rhabdomere were also present in deprived photoreceptors. We suggest that the elaboration of smooth endoplasmic reticulum as myeloid bodies in chromophore-deprived photoreceptors may stem from the hypertrophy of a biochemical system for processing the chromophore or the interruption of the intracellular pathway that normally carries visual pigment to the rhabdomere.     

Teratogenicity of retinoic acid and its effects on TGF-β2 expression in the developing cerebral cortex of the rat

Vitamin A metabolites are potent teratogens in a wide variety of species, including man. Transforming growth factor betas (TGF-s) are involved in several mammalian prenatal developmental processes. The aim of this study was to determine the effects of exogenous and excessive all-trans retinoic acid on TGF2 expression in the developing cerebral cortex of the rat. Many of the malformations including exencephaly, exophtalmus, abdominal wall defects, extremity reduction defects observed in this study were dependent on the time of administration of retinoic acid. TGF-2 was diversely expressed, as revealed immunohistochemically, in the cerebral cortex and plexus choroideus. The diversity depended on the gestational day and the was affected by the administration of retinoic acid. In the 15-day-old fetus from mothers who had been fed by gavage a single dose of 60mg/kg body weight of all-trans retinoic acid on the 8th day of gestation, TGF-2 immunoreactivity in the brain was decreased. However, by the 18th day of gestation, TGF-2 expression increased. The expression of TGF-2 in fetuses whose mothers had been given all-trans retinoic acid after the neurulation period (on day 12 of gestation) was generally similar to that in a control group. We conclude that all-trans retinoic acid leads to severe congenital malformations if administered before neurulation whereas if given after neurulation, it is not so teratogenic. Further, retinoic acid has a variable effect on the expression of TGF-2.     

Male swarms at landmarks and scramble competition polygyny inPolistes gallicus (Hymenoptera: Vespidae)

At the end of summer, males of Polistes gallicusfly in swarms around vertical landmarks and land in clusters on their favorite perches, where they drag their legs and abdomen. Here males occasionally crowd around a perched female; they make no effort to defend an exclusive mating territory but instead attempt to copulate by displacing rivals from the female. In this work we describe this spatial-nuptial system, which entails site fidelity without territoriality, unisexual swarms, common patrol routes, collective sexual approaches, and scramble competition polygyny. Mating success is evaluated in relation to the familiarity with flight paths (routine patrollers versus newcomers), to the type of sexual approach (single males versus in- group males), and, in the laboratory, to the individual activity level.     

Organ-specific crystalline structures of ferritin cores inβ-thalassemia/hemoglobin E

Summary The cores of ferritins isolated from different organs of human subjects with-thalassemia/hemoglobin E (-thal/HbE) disease have different size distributions and crystallinities depending on the source organ. These patients have not been treated by hypertransfusion regimen or iron chelation therapy.-Thal/HbE spleens and livers yield ferritin cores which are less crystalline than those isolated from normal spleens and livers, reflecting the more rapid deposition of iron in the diseased state. Ferritins isolated from the hearts and pancreases of-thal/HbE subjects were found to have larger, more crystalline cores than those from the-thal/HbE livers and spleens, possibly as a consequence of the role of the heart and pancreas as long-term iron deposition sites in this iron overload pathology.     

On the role of the invariant glutamine at position 54 in the human choriogonadotropin β subunit

The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone subunits from a variety of mammalian species. -Gin-54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single AG mutation in the LH gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH, with Arg replacing Gin-54, associated with the subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine , and the media were analyzed for holoproteins, which were characterizedin vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG(Glu-54) bound to almost as well as hCG wild-type, and the resulting heterodimer competed with [¹²⁵l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED₅₀s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG(Lys-54) associated very poorly with . Our results suggest that while Gin-54 in hCG participates in receptor binding, its major function appears to involve binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding.     

Free energies of amino acid adsorption on silica in neutral aqueous medium as estimated from high-performance liquid-chromatographic retention data

Summary Equilibrium constants (K) and free energies (–G) of amino acid adsorption on silica in a neutral aqueous medium were calculated from the retention values measured by means of high-performance liquid chromatography on a silica gel column. For most amino acids (with the exception of proline) –G values were negative andK < 1, thus showing very low adsorption. Influence of the structure of the-substituent on adsorbability is analyzed. A linear dependence of –G on the number of aliphatic carbon atoms was shown for the series: glycine-alanine-valine-leucine-isoleucine.Abbreviations Gly glycine - Ala alanine - Pro proline - Val valine - Ile isoleucine - Leu leucine - Ser serine - Thr threonine - Cys cysteine - Asn asparagine - Gln glutamine - Asp aspartic acid - Glu glutamic acid - Met methionine - His histidine - Phe phenylalanine - Tyr tyrosine - DOPA 3,4-dioxyphenylalanine - Trp tryptophan     

Construction of stable lactose-utilizing Xanthomonas campestris by chromosomal integration of cloned lac genes using filamentous phage ϕLf DNA

Summary Previously, we constructed a lactose-utilizing strain of Xanthomonas campestris, Xc17 (pKMLT), by cloning lacZY genes with the RK2-derived vector pLAFR1. In this study, the narrow-host-range, -galactosidase expression plasmid pKM was fused with an integration vector pS19 to form pSF14. Following insertion into Xc17, pSF14 was integrated into the host chromosome. The integration function was provided by the 0.85-kb EcoRI-PstI fragment from the filamentous phage Lf. The integration caused no adverse effect to the cells and was stable for at least 66 generations without selection. The engineered strain, Xc17::pSF14, was able to grow as well and produce as much xanthan gum in lactose medium as the wild-type cells did in glucose medium, and the Xc17(pKMLT) in lactose medium. Therefore, Xc17::pSF14 is potentially useful for xanthan production by direct use of whey lactose as the fermentation substrate. This study has advanced one more step our efforts to contruct lactose-utilizing X. campestris and confirmed the feasibility of using pS19 as an integration vector.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

A study of the vascular arrangement in the rat adrenal gland using non-radioactive microspheres

Summary The intra-glandular vascular arrangement in the adrenal has been studied using non-radioactive microspheres injected by three different routes: in-vivo injection into the left ventricle under pentobarbital anesthesia, postmortem orthograde, and postmortem retrograde injection. The doses of microspheres were 10⁵ (average size 24.7 m), 10⁶ (15.8 m) and 10⁷ (9.9 m). The entrapment rate of microspheres by the medulla as compared with the whole gland was measured in the serially sectioned tissue (section thickness 60 m).The entrapment rates of 25-m microspheres differed between the orthograde and retrograde injections, while the entrapment rates for 15-m microspheres were essentially similar irrespective of the route of injection.Our results support the conclusion from previous microangiographic studies that the adrenal cortex and medulla are supplied by different arteries but have a common venous outflow, and that direct communication between cortical and medullary sinusoids is not likely. The medullary blood flow per gram tissue weight is estimated to be larger than cortical blood flow.     

Der Tryptophanabbau bei Rhizobium leguminosarum

Summary The degradation of tryptophan (Try) and some of its potential intermediates has been studied in nodule bacteria (Rhizobium leguminosarum Frank, ATCC 10324). In feeding experiments with washed suspensions the following degradation products of Try could be identified by thin-layer chromatography: indolyl-3-pyruvic acid (IBS); indolyl-3-acetic acid (IES); -(indolyl-3)-lactic acid (IMS); indole-carboxylic acid-(3) (ICS); -(indolyl-3)-ethanol (-IÄ); indole-aldehyde-(3) (IAld); indolyl-3-acetaldehyde (IAAld); N-acetyl-tryptophan (Ac-Try).An active Try-transaminase leading to the formation of IBS has been demonstrated. Phenylpyruvic acid as well as -ketoglutaric acid served as amino group acceptors.The breakdown of Try was followed quantitatively by using C-14_(2-alanyl-) D,L-tryptophan. After 16 hrs nearly 16% of the original radioactivity was found in the ether-extractable material. IES and IMS were formed in much the highest concentrations.Indole-3-acetonitrile (IAN), although not a Try-metabolite in Rhizobium leguminosarum was converted to IES via indole-3-acetamide (IAAm). The following physiological pathways in the breakdown of Try in Rhizobium leguminosarum have been confirmed: Try Ac-Try and IBS; IBS IAAld; IAAld -IÄ and IES; no further degradation of IES was observed.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.