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).     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997     

Electron paramagnetic resonance saturation studies of P-700+ reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T1, and spin-spin, T2, relaxation times of P-700+ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K less than or equal to T less than or equal to 100 K. T1 was 200 mus at 100 K and increased to 900 mus at 10 K. T2 was 40 ns at 40 K and increased to 100 ns at 10 K. T1 for 40 K less than or equal to T less than or equal to 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T1 deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T1 of P-700+ were examined: T1 of P-700+ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700+ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700+ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700+ due to either the iron sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700+ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of P-700+ signal. The absence of dipolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700+ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

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.     

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.     

Nucleic acid relationships among Acholeplasma species.

3H-labeled Acholeplasma DNA probes were generated in vitro by the nick-translation method and used to determine the nucleotide sequence homology among the type strains of the eight currently recognized species of Acholeplasma. Very little nucleotide sequence homology (less than or equal to 18%) was found among the eight species, with heteroduplexes showing at least 12% or more mismatching as determined by thermal elution midpoints. The small amount of nucleotide sequence homology among the eight species indicates that these species are quite distinct and are not closely related to each other genomically.     

Antibody responses to galectin-8, TARP and TRAP1 in prostate cancer patients treated with a GM-CSF-secreting cellular immunotherapy

A critical factor in clinical development of cancer immunotherapies is the identification of tumor-associated antigens that may be related to immunotherapy potency. In this study, protein microarrays containing >8,000 human proteins were screened with serum from prostate cancer patients (N = 13) before and after treatment with a granulocyte–macrophage colony-stimulating factor (GM-CSF)-secreting whole cell immunotherapy. Thirty-three proteins were identified that displayed significantly elevated (P ≤ 0.05) signals in post-treatment samples, including three proteins that have previously been associated with prostate carcinogenesis, galectin-8, T-cell alternative reading frame protein (TARP) and TNF-receptor-associated protein 1 (TRAP1). Expanded analysis of antibody induction in metastatic, castration-resistant prostate cancer (mCRPC) patients (N = 92) from two phase 1/2 trials of prostate cancer immunotherapy, G-9803 and G-0010, indicated a significant (P = 0.03) association of TARP antibody induction and median survival time (MST). Antibody induction to TARP was also significantly correlated (P = 0.036) with an increase in prostate-specific antigen doubling time (PSADT) in patients with a biochemical (PSA) recurrence following prostatectomy or radiation therapy (N = 19) from in a previous phase 1/2 trial of prostate cancer immunotherapy, G-9802. RNA and protein encoding TARP and TRAP1 was up-regulated in prostate cancer tissue compared to matched normal controls. These preliminary findings suggest that antibody induction to TARP may represent a possible biomarker for treatment response to GM-CSF secreting cellular immunotherapy in prostate cancer patients and demonstrates the utility of using protein microarrays for the high-throughput screening of patient-derived antibody responses.