Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2

Two small plasmid RK2 derivatives, pSV6 and pSV16, were constructed and used for the isolation and characterization of trfA mutants temperature-sensitive (ts) for replication in Escherichia coli. Four of the mutants were examined for their ability to initiate replication from the RK2 replication origin in E. coli when present in cis with respect to the origin and in trans when present on a multicopy pBR322 replicon. Each of the mutant trfA genes exhibited temperature-sensitivity in supporting replication from the RK2 origin when present in cis, and the lowest nonpermissive temperature varied depending on the mutant. When the mutant trfA genes were present on the multicopy replicon (in trans), three of the four mutant genes could support replication of the RK2-oriV plasmid pSV16 at all temperatures tested. However, with the exception of one of the mutants, the activity was reduced when compared to wild-type. The increased activity in trans possibly is the result of the increased cellular level of the TrfA protein when compared with the in cis situation where the mutant trfA gene is at a much lower copy-number. Two of the mutants also were tested in cis for temperature sensitivity in Pseudomonas aeruginosa. One of the mutants did not exhibit temperature sensitivity under the conditions employed. The second mutant showed some temperature sensitivity but the nonpermissive temperature pattern was different than that found in E. coli.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Is hypothermia necessary for the winter survival of the GoldcrestRegulus regulus?

Summary The Goldcrest is an interesting species for studies of physiological and behavioural adaptions to cold, since it is the smallest species present in Europe. Although many small birds have to lower their metabolic rate and enter hypothermia in order to make their energy reserves last throughout the night, our measurements of oxygen consumption of five Goldcrests suggest that these birds may be able to balance their nocturnal energy budget during the winter at normothermic body temperatures, provided that the available food supply is adequate. These results are in agreement with those of another small bird, the Common Bushtit, which, like the Goldcrest, reduce nocturnal heat loss by contact roosting. Since the energy saved by hypothermia decreases with decreasing ambient temperature and also decreasing body weight, this strategy might not be such an advantage for the smallest birds living at very low ambient temperatures.

---

Zusammenfassung Am Wintergoldhähnchen als dem kleinsten europäischen Vogel interessieren besonders seine physiologischen und Verhaltens-Anpassungen an niedere Temperaturen. Viele Kleinvögel senken ihre Stoffwechselaktivität und verfallen in Hypothermie, um mit ihren Energie-Reserven während der langen Winternächte auszukommen. Unsere Untersuchungen über den O₂-Verbrauch von 5 Wintergoldhähnchen legen jedoch nahe, daß sich das nächtliche Energie-Budget auch im Tiefwinter auf Normalniveau bewegt und daß sie bei ausreichendem Nahrungsangebot ihre normale Körpertemperatur auch nachts beibehalten. Ähnliche Befunde liegen von den vergleichbaren kleinen amerikanischen Buschmeisen vor, die wie Goldhähnchen den nächtlichen Wärmeverlust durch Schlafen auf engem Gefiederkontakt reduzieren. Es ist anzunehmen, daß die Hypothermie als Überwinterungsstrategie für unsere kleinsten Vögel garnicht so vorteilhaft wäre: Die durch Hypothermie eingesparte Energie geht mit abnehmender Umgebungstemperatur und mit geringerer Körpergröße wieder verloren.

     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [³H]retinol and ¹⁴C-labelled vitamin D-3, ¹⁴C-labeled vitamin D-3 appeared later in the intestinal lymph than [³H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Discrimination between Weaned and Unweaned Atlantic Cod (Gadus morhua) in Capture-Based Aquaculture (CBA) by X-Ray Imaging and Radio-Frequency Metal Detector

The aim of this study was to investigate the feasibility of two detection methods for use in discrimination and sorting of adult Atlantic cod (about 2 kg) in the small scale capture-based aquaculture (CBA). Presently, there is no established method for discrimination of weaned and unweaned cod in CBA. Generally, 60–70% of the wild-caught cod in the CBA are weaned into commercial dry feed. To increase profitability for the fish farmers, unweaned cod must be separated from the stock, meaning the fish must be sorted into two groups – unweaned and weaned from moist feed. The challenges with handling of large numbers of fish in cages, defined the limits of the applied technology. As a result, a working model was established, focusing on implementing different marking materials added to the fish feed, and different technology for detecting the feed presence in the fish gut. X-ray imaging in two modes (planar and dual energy band) and sensitive radio-frequency metal detection were the detection methods that were chosen for the investigations. Both methods were tested in laboratory conditions using dead fish with marked feed inserted into the gut cavity. In particular, the sensitive radio-frequency metal detection method with carbonyl powder showed very promising results in detection of marked feed. Results show also that Dual energy band X-ray imaging may have potential for prediction of fat content in the feed. Based on the investigations it can be concluded that both X-ray imaging and sensitive radio-frequency metal detector technology have the potential for detecting cod having consumed marked feed. These are all technologies that may be adapted to large scale handling of fish from fish cages. Thus, it may be possible to discriminate between unweaned and weaned cod in a large scale grading situation. Based on the results of this study, a suggestion for evaluation of concept for in-situ sorting system is presented.     

Conjugative transfer of the naturally occurring plasmids of Acetobacter xylinum by IncP-plasmid-mediated mobilization.

Broad-host-range plasmids and cloning vectors were conjugatively transferred to Acetobacter xylinum. One of the plasmids, RP4::Mu cts61, was used for the insertion of Tn1 into the 16-, 44-, and 64-kilobase-pair plasmids of A. xylinum. The Tn1-labeled plasmids could be mobilized by a helper plasmid. Many of the Tn1 insertions affected the copy number of the plasmids.     

Oxidative damage to purines in DNA: role of mammalian Ogg1

DNA damage caused by reactive oxygen species is ubiquitous to all living organisms. More than 60 different base lesions have been identified, and the majority of these are removed via the base excision repair pathway. This pathway appears to represent a highly conserved and ancient mechanism of defence counteracting spontaneous DNA decay. In this review, we describe in more detail the Ogg1 enzyme and its conserved action of removing the oxidised base, 7,8-dihydro-8-oxoguanine (oxo(8)G). Recent updates include the cancer-prone ogg1/myh double knockout mouse and an elegant study which looks at the ability of hOgg1 to distinguish between the mutagenic lesion, oxo(8)G, and the vast majority of normal bases.     

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.     

What Stakeholders Think About Marine Protected Areas: Case Studies from Spain

Marine protected areas (MPAs) are often met with reluctance by affected stakeholders, and in some instances outright objection. Some argue that this is due to insufficient understanding of the functions of MPAs. Others suggest that it could be because of a perception that they are losing more than they are gaining. It is also possible that stakeholders are generally supportive of the idea but think that the MPA should be located elsewhere. We argue that it is images people have about what the MPA is and does that determine how they react. Drawing from three MPA case studies in Spain, we illustrate the importance of critical examination of stakeholders’ images and what they imply for the governance of MPAs.