Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli

A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step.     

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

The effects of serotonergic and adrenergic receptor antagonist on prolactin release in the monkey.

The influence of serotonergic and adrenergic antagonists on serum prolactin levels was studied in ketamine anesthetized monkeys. Methysergide, a serotonergic receptor blocker, at 0.035, 0.1 and 1 mg/kg body weight induced a rapid and transient increase in serum prolactin. Cyproheptadine, another serotonergic receptor blocker, at 0.05, 0.5 and 1 mg/kg induced a rapid and sustained increase in serum prolactin. SQ 10631, a third serotonergic receptor blocker, had a minimal effect on increasing basal prolactin levels even at doses as high as 10 mg/kg. Propranolol, a β adrenergic blocker, at a dose of 5 mg/kg induced a small sustained increase in serum prolactin, while a lower dose (1 mg/kg) had a slight but significant effect. Phentolamine, an α adrenergic receptor blocker, at a dose of 5 mg/kg induced a rapid and transient increase in plasma prolactin while a lower dose (1 mg/kg) had no effect. Phenoxybenzamine, a potent α adrenergic receptor blocker, had only a minimal effect on prolactin release even at doses of 3 and 5 mg/kg. It appears that the time course and extent of prolactin release differs among neural antagonists even within the same biogenic amine system.     

Definition of the boundaries of the origin of DNA replication in simian virus 40.

We have determined by use of DNA sequencing techniques the exact location of the deletion in d1 892, a viable deletion mutant of Simian virus 40 (SV40) reported to map very near the unique replication origin or SV40. With the help of this localization we have narrowed down the boundaries of the replication origin to 85 nucleotides within the sequence of SV40.     

Structure of guanylyl-3',5'-cytidine monophosphate. II. Description of the molecular and crystal structure of the calcium derivative in space group P2(1).

The structural features of calcium guanosine-3′,5′-cytidine monophosphate (GpC) have been elucidated by X-ray diffraction analysis. The molecule was crystallized in space group P2₁ with cell constants of a = 21.224 Å, b = 34.207 Å, c = 9.327 Å, and β = 90.527°, Z = 8. The hydration of the crystal is 21% by weight with 72 water molecules in the unit cell. The four GpC molecules in the asymmetric unit occur as two Watson-Crick hydrogen-bonded dimers related by a pseudo-C face centering. Each dimer consists of two independent GpC molecules whose bases are hydrogen bonded to each other in the traditional Watson-Crick fashion. Each dimer possesses a pseudo twofold axis broken by a calcium ion and associated solvent. The four molecules are conformationally similar to helical RNA, but are not identical to it or to each other. Instead, values of conformational angles reflect the intrinsic flexibility of the molecule within the range of basic helical conformations. All eight bases are anti, sugars are all C3′-endo, and the C4′-C5′ bond rotations are gauche-gauche. The R factor is 12.6% for 2918 observed reflections at 1.2-Å resolution.     

On the mass distribution model for microbial cell populations

The full implications of a statistical model for growth of a microbial cell population using cell mass as the index of physiological state have been examined by solving the partial differential integral equations resulting from the model. Calculations reveal that a lag phase is predicted during the initial stages of batch growth although no specific cellular mechanism for the phenomenon of lag had been incorporated into the model. The model predicts several situations of batch and continuous growth in which the population density and biomass concentration show opposing trends due to significant variation in the cell mass distribution with time.