Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.     

Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney

Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1-3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.     

Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. "Leaky" cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.     

Interaction of the bisbiguanides chlorhexidine and alexidine with phospholipid vesicles: evidence for separate modes of action

Strains of Providencia stuartii with demonstrated resistance towards chlorhexidine did not show such resistance towards either of the related biguanide antiseptics, alexidine or vantocil. Alexidine promoted a significantly faster alteration in the permeability of Escherichia coli cell membranes towards various metal cations than chlorhexidine. Differential thermal analysis of various mixed lipid vesicles and pure phospholipid vesicles showed alexidine to share with vantocil the property of producing lipid phase separation and domain formation. It is suggested that the nature of the end-group on the biguanides affects the ability to produce lipid domains in cell membranes and this this might, in turn, affect activity and resistance patterns observed.     

Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells.

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.     

Actigraphy: A Means of Assessing Circadian Patterns in Human Activity

-Twenty-three diurnally active (0705-2333), healthy persons between 22 and 54 yrs of age and without history of sleep abnormality were monitored continuously for 120 consecutive hr (five days) by wrist actigraphy. Circadian rhythms of high amplitude were detected by cosinor analysis for each participant and for the groups of 10 males and 13 females with the average span of heightened activity timed between ∼1330 and 1605. The circadian peak-trough difference in wrist movement was marked, equalling aproximately 75% of the 24-hr mean level. In 19 of 23 participants, the 24-hr mean of wrist activity varied between 140-180 movements/min, with four persons exhibiting lesser means of 110-140 movements/min. With respect to the daytime span of activity, the mean wrist movement of individual participants ranged from 155-265 movements/min, with the majority (20/23) varying between 185-245 movements/min. During nocturnal sleep the mean wrist activity level was quite low, varying between individuals from 5 to 25 movements/min for 21 of 23 persons. Wrist actigraphy proved to be well-accepted and was a most reliable means of monitoring aspects of body movement during activity and sleep in ambulatory persons adhering to usual life habits and pursuits.     

Chemical synthesis, cloning and expression in mammalian cells of a gene coding for human tissue-type plasminogen activator

A 1610-bp DNA duplex coding for human tissue-type plasminogen activator has been chemically synthesized using the phosphoramidite procedure, adapted for a custom-built gene synthesizer. The synthesizer, which was designed for both simplicity and speed, permits the rapid construction of relatively large genes and compares favorably in speed with alternative cDNA isolation procedures. The plasminogen activator gene has been expressed in mammalian cells and shown to produce authentic protein by an immuno-activity assay.     

In vivo brown fat response to hypothermia and norepinephrine in the ovine fetus

The goal of this study was to assess the response of fetal brown fat in vivo to hypothermia and norepinephrine infusion. In 10 unanaesthetized, chronically-prepared fetal sheep (133 +/- 2 days of gestation) cold water was passed through tubing encircling the fetus in utero and plasma glycerol concentration was measured as an indicator of brown fat activity. Following cooling for 60 min, amniotic fluid temperature fell 7.79 degrees C to 31.66 +/- 1.73 degrees C (n = 8, P less than 0.001) and maternal temperature fell 0.63 degree C to 38.63 +/- 0.08 degrees C (n = 9, P less than 0.001). Eight of the fetuses were subjected to a second experiment in which norepinephrine was infused intravenously for 15 min. During infusion fetal arterial temperature fell 0.38 degrees C to 39.05 +/- 0.25 degrees C (n = 7, P less than 0.05). Amniotic fluid temperature (n = 7, NS) and maternal arterial temperature (n = 7, NS) remained constant. Glycerol concentration during the infusion increased from 0.73 to 1.27 mg/dl, a 74% increase over control (n = 8, P less than 0.001). Although clearly detectable, these glycerol responses to hypothermia and norepinephrine stimulation are one-third or less of those achieved after birth, indicating that thermogenesis remains quiescent in the near-term fetal sheep, despite powerful stimuli for activation.     

Polyamines Differentially Inhibit Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

The effects of the naturally occurring polyamines spermine and spermidine on phosphorylation promoted by cyclic AMP (cAMP)-dependent protein kinase (PK) (cAMP-PK; EC 2.7.1.37) were studied using the brain of the tobacco hornworm, Manduca sexta. Four particulate-associated peptides (280, 34, 21, and 19 kilodaltons) in day 1 pupal brains are endogenous substrates for a particulate type II cAMP-PK. These phosphoproteins are present in brain synaptosomal, as well as microsomal, particulate fractions but are not present in the cytosol. They are distributed throughout the CNS and PNS and are present in several nonneuronal tissues as well. Phosphorylation of these proteins via cAMP-PK was inhibited markedly by micromolar concentrations of spermine and spermidine. Other particulate-associated peptides phosphorylated via a Ca2+/calmodulin-PK or Ca2+ and cAMP-independent PKs were unaffected by polyamines, whereas the phosphorylation of a 260-kilodalton peptide was markedly enhanced. Spermine did not exert its inhibitory effect indirectly by enhancement of cAMP or ATP hydrolysis or via proteolysis, but its action appears to involve a substrate-directed inhibition of cAMP-PK-promoted phosphorylation as well as enhanced dephosphorylation. Although addition of spermine resulted in marked ribosome aggregation in synaptosomal and microsomal particulate fractions, this phenomenon was not involved in the inhibition of cAMP-PK-promoted phosphorylation.