Growth-induced buckling of an epithelial layer

We use a proof-of-concept experiment and two mathematical models to explore growth-induced tissue buckling, as may occur in colorectal crypt formation. Our experiment reveals how growth of a cultured epithelial monolayer on a thin flexible substrate can cause out-of-plane substrate deflections. We describe this system theoretically using a ‘bilayer’ model in which a growing cell layer adheres to a thin compressible elastic beam. We compare this with the ‘supported-monolayer’ model due to Edwards and Chapman (Bull Math Biol 69:1927–1942, 2007) for an incompressible expanding beam (representing crypt epithelium), which incorporates viscoelastic tethering to underlying stroma. We show that the bilayer model can exhibit buckling via parametric growth (in which the system passes through a sequence of equilibrium states, parameterised by the total beam length); in this case, non-uniformities in cell growth and variations in cell–substrate adhesion are predicted to have minimal effect on the shape of resulting buckled states. The supported-monolayer model reveals how competition between lateral supports and stromal adhesion influences the wavelength of buckled states (in parametric growth), and how non-equilibrium relaxation of tethering forces influences post-buckled shapes. This model also predicts that non-uniformities in growth patterns have a much weaker influence on buckled shapes than non-uniformities in material properties. Together, the experiment and models support the concept of patterning by growth-induced buckling and suggest that targeted softening of a growing cell layer provides greater control in shaping tissues than non-uniform growth.     

γ-Herpesvirus reactivation differentially stimulates epitope-specific CD8 T cell responses

The γ-herpesviruses are characterized by their ability to establish lifelong latency. Subsequent immune suppression leads to viral reactivation from latency and the onset of a variety of pathologies, including lymphoproliferative disease and cancers. CD8 T cells play a key role in preventing reactivation of latent virus. Therefore, to develop effective therapeutic immune strategies, it is essential to understand the maintenance of CD8 T cell responses during latency. Because the γ-herpesviruses are highly species-specific and mice cannot be infected with the human pathogens, EBV or Kaposi's sarcoma-associated herpesvirus, we have used a natural rodent γ-herpesvirus experimental infection model, γ-herpesvirus-68. In this report, we show that during long-term latent infection, naive CD8 T cells are recruited into the ongoing immune response in an epitope-specific manner. When virus reactivation is induced in vivo, the recruitment of CD8 T cells for some, but not all, epitopes is enhanced. The variation in recruitment is not due to differences in epitope presentation. We also show that CD8 T cells that are newly stimulated during reactivation are functionally impaired compared with acutely stimulated cells in terms of cytokine production. Thus, our results demonstrate unexpected complexity in the response of CD8 T cells specific for different viral epitopes that were stimulated during acute infection, quiescent latency, and reactivation.     

Genetic regulation of fluxes: iron homeostasis of Escherichia coli

Iron is an essential trace-element for most organisms. However, because high concentration of free intracellular iron is cytotoxic, cells have developed complex regulatory networks that keep free intracellular iron concentration at optimal range, allowing the incorporation of the metal into iron-using enzymes and minimizing damage to the cell. We built a mathematical model of the network that controls iron uptake and usage in the bacterium Escherichia coli to explore the dynamics of iron flow. We simulate the effect of sudden decrease or increase in the extracellular iron level on intracellular iron distribution. Based on the results of simulations we discuss the possible roles of the small RNA RyhB and the Fe–S cluster assembly systems in the optimal redistribution of iron flows. We suggest that Fe–S cluster assembly is crucial to prevent the accumulation of toxic levels of free intracellular iron when the environment suddenly becomes iron rich.     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

Synthesis of novel hydroxymethyl substituted analogues related to carbovir and neplanocin A

Two enantiomerically pure hydroxymethyl substituted cyclopentene nucleoside analogues (42 and 53) related to carbovir and neplanocin A, respectively, were prepared from the chiral pool of iridoid glucosides. In addition two saturated hydroxymethylated analogues (44 and 45) were obtained from a protected intermediate.     

Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini. Formation of CrkII-protein complexes.

Crk belongs to a family of adapter proteins whose structure allows interaction with tyrosine-phosphorylated proteins and is therefore an important modulator of downstream signals, representing a convergence of the actions of numerous stimuli. Recently, it was demonstrated that cholecystokinin (CCK) induced tyrosine phosphorylation of proteins related to fiber stress formation in rat pancreatic acini. Here, we investigated whether CCK receptor activation signals through CrkII and forms complexes with tyrosine-phosphorylated proteins in rat pancreatic acini. We demonstrated that CCK promoted the transient formation of CrkII-paxillin and CrkII-p130Cas complexes with maximal effect at 1 min. Additionally, CCK decreased the electrophoretic mobility of CrkII. This decrease was time- and concentration-dependent and inversely related with its function. Carbachol and bombesin also decreased CrkII electrophoretic mobility, whereas epidermal growth factor, vasoactive intestinal peptide, secretin or pituitary adenylate cyclase-activating polypeptide had no effect. CCK-induced CrkII electrophoretic shift was dependent on the Src family of tyrosine kinases and occurred in the intact animal, suggesting a physiological role of CrkII mediating CCK actions in the exocrine pancreas in vivo.     

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

Response to different sources of vitamin E orally injected and to various doses of vitamin E in calf starter on the plasma vitamin E level in calves around weaning

Calves often face a lower plasma vitamin E level than the recommended level (3 µg/ml for adult cows) after weaning, a level which has been related to a good immune response. Two experiments were performed to determine the most effective source and level of vitamin E to be included in a calf starter to maintain the plasma vitamin E level above the recommended level after weaning. Experiment 1 (Exp 1) and experiment 2 (Exp 2) included a total of 32 and 40 calves, respectively, from 2 weeks before weaning until 2 weeks after weaning. In Exp 1, calves were orally injected a daily dose of different vitamin E sources including, no α-tocopherol (0 dose; Control), 200 mg/d of RRR-α-tocopherol (ALC), 200 mg/d of RRR-α-tocopheryl acetate (ACT), or 200 mg/d of all-rac-α-tocopheryl acetate (SYN). In Exp 2, a dose response study was carried out with 0, 60, 120, and 200 mg/kg of ALC in a pelleted calf starter. Final BW (100 ± 16 and 86 ± 11 kg) and average daily gain (956 ± 303 and 839 ± 176 g/d in Exp 1 and 2, respectively; mean ± SD) were unaffected by either source or level of α-tocopherol. In Exp 1, the plasma RRR-α-tocopherol level was affected by α-tocopherol source (P < 0.001), week (P < 0.001), and interaction between them (P < 0.001). At weaning time, the plasma RRR-α-tocopherol was 2.7, 2.1, 1.1, and 0.8 μg/ml in ALC, ACT, SYN, and Control, respectively. In Exp 2, the plasma α-tocopherol level was affected by ALC dose (P = 0.04), week (P < 0.001), and a tendency for an interaction between them was observed (P = 0.06). At weaning, a 36, 31, and 28% reduction in plasma α-tocopherol level was observed compared to the beginning of the experiment with 0, 60, and 120 mg/kg of ALC, respectively; however, with 200 mg/kg of ALC, a 9% increase in the plasma α-tocopherol level was observed. In addition, 200 mg/kg of ALC was able to maintain plasma α-tocopherol after weaning higher than the recommended level. The results showed that the ALC was the most efficient source of α-tocopherol supplementation to be used in a calf starter. In addition, the 200 mg/kg of ALC in the calf starter was the only effective dose to maintain the postweaning plasma vitamin E concentration at the recommended level after weaning and α-tocopherol similar to that observed before weaning.     

Structural difference at the active site of dibucaine resistant variant of human plasma cholinesterase.

Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The esterases were labeled with diisopropyl-1, 3-C14-fluorophosphate (DFP) aminoethylated, and digested by trypsin. The trytic digests were subjected to high voltage electrophoresis, and the radioactive peptides were detected by radioautography. Comparison of the peptides revealed different electrophoretic mobilities of the usual and atypical (dibucaine resistant) plasma cholinesterase peptides. The results are consistent with a structural abnormality of the active center in the variant enzyme. No difference was observed an the esteratic site of the enzyme with C5 component.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.