The thickness of the mucus layer in different segments of the rat intestine

Summary The thickness of the pre-epithelial mucus layer has been measured in different gut segments of rats kept under normal (ad libitum) feeding conditions, and after 48 h of fasting, using cryostat sections and celloidin stabilization from samples containing luminal contents. The mucus layer of the stomach, duodenum, jejunum, ileum, caecum, proximal colon, colon transversum, distal colon and rectum was studied in five groups of male rats (10, 40, 70 and 150 days of age, and older). Underad libitum feeding conditions, a distinct and continuous mucus layer, with a thickness of more than 3 μm, was only observed in the colon transversum, in the distal colon, in the rectum and in the stomach. No pre-epithelial mucus layer was observed in the duodenum and jejunum where the glycocalix from the apical membrane of the superficial cells appeared to be in a direct contact with the luminal ingesta. In the ileum, caecum and the proximal colon, the surface epithelium of the mucosa was only partly covered by a mucus layer of highly variable thickness. After 48 h of fasting, a mucus layer of 28.8 ± 25.6 μm and 93.3 ± 59.4 μm thickness, respectively, was found in the duodenum and jejunum of adult rats, but no increase in the thickness of the mucus layer was observed in the rat hind gut.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ecological correlates of hind-limb length in the Carnivora

What determines the lengths and proportions of mammalian limbs? While the answer to this question is still largely unknown, a number of workers have recently begun analysing the selection of morphology in a rigorous framework, searching for quantitative links between structure, performance, and their ecological and behavioural context. The present study investigates a variety of ecological and behavioural variables to determine whether or not they are correlated with hind-limb length in the Carnivora. Data were analysed by using phylogenetically independent contrasts and phylogenetic analysis of covariance. We found that traditional perceptions of limb length are often inaccurate; some species widely regarded as relatively long-legged actually have limb lengths near those expected for their body size. Interestingly, relative hind-limb length is not a significant predictor of distance moved daily, home-range area, or prey size. Phylogenetic ANCOVA results, however, indicate a relationship between prey-capture behaviour and relative hind-limb length. These findings suggest that the evolution of carnivoran limb length has been most influenced by selection for prey-capture behaviour. These results, coupled with those of other studies, can be used to suggest which performance variables could most fruitfully be studied in the laboratory to understand the selection of the structure of the mammalian limb. We suggest that relevant performance variables might be: maximum jump height and/or length, the ability to generate outforces, and levels of stress tolerance in limb bones during prey pursuit/capture.     

Retrofitting YACs for direct DNA transfer into plant cells

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

Posttetanic potentiation of human dorsiflexors

O'Leary, Deborah D., Karen Hope, and Digby G. Sale.Posttetanic potentiation of human dorsiflexors.J. Appl. Physiol. 83(6):2131-2138, 1997.Twitch contractions of the ankle dorsiflexors were evoked before and after applied 7-s tetanic stimulation at 100 Hzin 20 young adults. Torque decreased 15% during the tetanus. At 5 safter tetanus, twitch peak torque had potentiated 45%. Potentiationdeclined to 28% after 1 min, rose slightly to 33% at 2 min, anddeclined slowly with potentiation still 25% after 5 min. There waslarge intersubject variation in the amount of potentiation(5-140%) and its persistence (5 to 20 min). The muscle compoundaction potential (M wave) did not change significantly (from pretetanicvalue) at 5 s after tetanus but increased sharply (26%) at 2 min andthen subsided. Twitch half relaxation time (23%) decreasedsignificantly more than twitch rise time (13%) 5 s after tetanus andrecovered more slowly. Twitch rates of torque development (75%) andrelaxation (71%) increased similarly 5 s after tetanus and were stillelevated (~25%) at 5 min. The extent of twitch torque potentiationwas significantly inversely correlated with pretetanic twitch rise time(r = 0.69), half relaxation time (r = 0.61), andtwitch-to-tetanus ratio (r = 0.66). The data indicate that posttetanic potentiation has agreater effect on twitch half relaxation time than on time to peaktorque and is more prominent in muscles with a short twitch time courseand small twitch-to-tetanus ratio.

     

Sex-related differences in first-stage impedance cardiographic variables

Impedance cardiography (ZCG), a noninvasive technique used to determine stroke volume and ventricular performance, is currently being utilized more extensively in psychophysiological research and may also prove a useful tool in the control of hypertension. To date, most studies employing ZCG have tested male subjects or patients. However, women and men differ on two important parameters used to calculate stroke volume: thoracic impedance (Zo), and the first derivative of change in impedance over time (dZ/dt_min). Examination of the clinical records of 19 females and 19 males, all nonmedicated, indicated that women had higher Zo levels (F(1, 36)=46.2,p<.0001) and higher dZ/dt_min levels (F(1, 36)=51.8, p<.0001), although calculated stroke volume indexes did not differ. A second sample of 10 women and 8 men, all healthy and nonmedicated, confirmed these findings. The differences in Zo are not due entirely to sex differences in height, weight, percent body fat, and subscapula skin-fold thickness, although these factors differ across sexes. It is important for researchers and clinicians utilizing ZCG with female subjects to be aware of these differences.