Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

Purine salvage as metabolite and energy saving mechanism in Camelus dromedarius: the recovery of guanine

The preservation of purine ring as purine bases appears to be a common feature of camel liver. Hepatic guanine appears to be actively converted into GMP in the camel rather than further degraded. The limiting step of guanine degradation appears to be the lack of hepatic guanase activity. Higher purine bases over uric acid ratios were found in camel urine with respect to those of zebu.     

Untersuchungen zur Pollenentwicklung und Pollenschlauchbildung bei höheren Pflanzen

Zusammenfassung In reifen Pollenkörnern der beiden Sommergerstensorten Amsel und Wisa sowie der F₁-Pflanzen, die aus den Sorten-Kreuzungen Impala X Wisa und Union X Wisa hervorgegangen sind, wurde die DNS-Menge der Kerne cytophotometrisch bestimmt. Die Messungen wurden zugleich bei Spermakernen und vegetativen Kernen eines Pollenkorns vorgenommen. Außerdem wurde der DNS-Gehalt von Kernen von Wurzelspitzen-Zellen der Sorten Amsel und Wisa ermittelt.Amsel und Wisa unterscheiden sich signifikant im DNS-Gehalt der Kerne von Wurzelspitzen-Zellen.Die Befunde der Messungen des DNS-Gehalts von vegetativen und Sperma-Kernen bei vier Gerstenformen zeigen, daß zum Zeitpunkt der Anthese die DNS-Replikationsphase bei vegetativen und Sperma-Kernen noch nicht abgeschlossen ist. Der DNS-Gehalt vegetativer Kerne von Wisa ist signifikant niedriger als die entsprechenden Werte der übrigen drei Gerstenformen. Der Verlauf der DNS-Replikation erfolgt bei beiden Spermakernen synchron. Hingegen verläuft die DNS-Replikation bei vegetativen und Sperma-Kernen mit großer Wahrscheinlichkeit nicht gleichsinnig.Im Diskussionsteil wird erstens erläutert, daß bei allen bisher analysierten Pflanzenarten des zwei- oder dreikernigen Pollenkorn-Typs zum Zeitpunkt der Pollenreife die DNS-Replikation der generativen bzw. Sperma-Kerne eingesetzt hat, aber je nach Pflanzenart noch nicht beendet sein muß. Zum gleichen Zeitpunkt der Pollenkornentwicklung kann der vegetative Kern in Abhängigkeit von der Pflanzenart auf dem C-Niveau verharren, eine teilweise oder bereits abgeschlossene DNS-Replikation erfahren haben oder schon teilweise oder ganz degeneriert sein, ohne zuvor eine DNS-Replikation vollzogen zu haben. Zweitens wird in diesem Abschnitt diskutiert, daß mit großer Wahrscheinlichkeit im Ablauf der DNS-Replikation zwischen zwei- und dreikernigen Pollenkorn-Typen keine Unterschiede bestehen. Drittens wird die Hypothese vertreten, daß nur auf einem sehr frühen Stadium die normale Pollenkornentwicklung einschließlich des Ablaufs der DNS-Replikation insbesondere des vegetativen Kerns so abgewandelt werden kann, daß aus Pollenkörnern haploide Pflanzen erzeugt werden können.

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The development of pollen grains and formation of pollen tubes in higher plantsIII. DNA-replication of vegetative and sperm nuclei in mature pollen grains of barley

Summary The DNA-content of vegetative and sperm nuclei in mature pollen grains of the barley varieties Amsel and Wisa and the F₁-plants of crossings of the barley varieties Impala X Wisa and Union X Wisa was determined by cytophotometry. In addition, the DNA-content of nuclei of root tips of Amsel and Wisa was cytophotometrically measured.The DNA contents of the nuclei in root tips of Amsel and Wisa differed significantly.The data obtained from the measurements of the vegetative and sperm nuclei of the four types of barley show that DNA-replication continues in the nuclei of mature pollen grains. The DNA values of vegetative nuclei of Wisa are significantly lower than the values of Amsel and of the F₁ plants. The DNA values of the different nuclei indicate that DNA replication of both types of sperm nuclei is synchronous, whereas it probably is not synchronous in vegetative and sperm nuclei respectively.In the discussion it is pointed out that a survey of the literature shows that in all of the plant species having binucleate or trinucleate pollen DNA replication of generative and of sperm nuclei has started at the time of pollen grain maturation. Depending on the plant species, replication may or may not be completed in the mature pollen grain. At a given stage of development of the pollen grain the vegetative nucleus may be arrested at the C-stage, may have partially or completely finished its DNA replication or may be partially or completely degenerated without prior replication of DNA.In the second part of the discussion it is stated that the course of DNA replication is likely to be similar in binucleate and trinucleate pollen grains. Thirdly, the hypothesis is discussed that in order to get haploid plants from pollen grains, changes in the normal development of the pollen grain and in the pattern of DNA replication must occur at a very early stage of pollen grain development.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Angenommen durch F. Mechelke

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Mein Dank grit Herrn Prof. Dr. F. Mechelke fiir die Anregung zu diesen Untersuchungen sowie fiir die Unterstfitzung und die kritischen Diskussionen w/ihrend ihres Verlaufs und Fr/iulein H. Nagel fiir zuverl/issige technische Hilfe.     

The role of the cysteine-rich region of the β2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

The cysteine-rich region (CRR) of the β2 integrin subunit was replaced by that of β1 to give the chimera β2NV1. β2NV1 can combine with αL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the β2 interaction with αL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing αLβ2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic β2 CRR.     

Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.     

Repair mechanisms involved in muscle regeneration following partial excision of the rat gastrocnemius muscle

The sequential cytological events of the regeneration process, after partial excision of the gastrocnemius muscle in the rat, were followed by light and electron microscopy. During the first 2 days after injury leukocytes and macrophages infiltrate into the traumatized area. Myogenic regeneration is then characterized by mainly two repair mechanisms. Mononucleated cells, that populate the excised area, most probably fuse together to give rise to newly formed multinucleated myotubes that further develop to striated myofibers. Another mechanism involves the repair of injured muscle fibers by the possible fusion of mononucleated cells with their necrotic cut ends. Consequently, by addition of nuclei and new muscular material, sarcoplasmic outgrowths from the injured fibers are formed. It is concluded that mainly two repair mechanisms are involved in the regeneration process following partial excision of a muscle: addition of new muscle fibers in a process similar to that of embryonic myogenesis and also meristic growth from the injured fibers.     

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)