Transfectability of rough strains of Salmonella typhimurium.

Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl2. The yield of infectious centers is about 10(-8) per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate-galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are not longer competent for transfection.     

Transfection of Staphylococcus aureus with Bacteriophage Deoxyribonucleic Acid

Staphylococcus aureus cells of strain 8325 (N) are competent for phage deoxyribonucleic acid (DNA) when harvested in the early exponential growth phase. Phenotypic expression of the competence requires divalent cations, and calcium ions are most effective. Treatment of phage DNA with deoxyribonuclease completely destroys infectivity and heat-denaturated DNA is not infectious. The highest frequency of transfection is around 10(4) plaque-forming units per mug of DNA.     

Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis.

An X-ray structure analysis of a crystal of mutant Asp229----Ala of cyclodextrin glycosyltransferase from Bacillus circulans (Ec 2.4.1.19) that had been shortly exposed to beta-cyclodextrin showed density corresponding to a maltose bound at the catalytic center. The crystal structure was refined to an R-factor of 18.7% at 2.5-A resolution. The catalytic center is defined by homology with the structurally known alpha-amylases and by the observation that mutants Asp229----Ala and Asp328----Ala are almost inactive. By model building, the density-defined maltose was extended to a full beta-cyclodextrin, which then indicated the general locations of seven subsites for glucosyl units. The catalytically competent residues Asp229, Glu257, and Asp328 are at the reducing end of the density-defined maltose. In the unligated wild-type structure, Glu257 and Asp328 form a 2.6-A hydrogen bond between their carboxylates in an arrangement that resembles those of the catalytically competent carboxylates in acid proteases. Presumably, the first catalytic step is an attack of the proton between Glu257 and Asp328 on the oxygen of the glycosidic bond.     

The nurse's role in immediate postoperative care.

From the time that a patient leaves the care of the anaesthetist after an operation until he wakes in the ward his physiological state should be continuously and expertly supervised. Postoperative nurses are provided only when the operating theatre has a recovery room. A survey among consultants and nurses in one region showed that many surgical units did not have recovery rooms and that inexperienced ward nurses were often sent to collect patients. The survey showed that most nurses were competent to care for unconscious patients so long as an emergency did not arise. In many hospitals the facilities for the safe nursing of postoperative patients were totally inadequate. The very least that is needed is good communications with the anaesthetist, adequate lighting, and a source of oxygen and suction. Because of the shortage of nurses likely to have to care for postanaesthetic patients early on and to train them accordingly. Nevertheless, recovery nurses, whose sole responsibility is to care for a patient until be has recovered from anaesthesia, should be appointed for all busy surgical units.     

Use of a light-induced respiratory transient to measure the optical cross section of photosystem I in chlorella

A method has been developed whereby the magnitude of a transient in O₂ uptake attributable to photosystem (PS) I activity, following single-turnover laser flashes of varying energy, can be used to measure the optical cross section of PSI. As measurements are made under the identical physiological conditions for which the cross section of PSII has previously been determined (AC Ley, DC Mauzerall 1982 Biochim Biophys Acta 680: 96-105), it is now possible to simultaneously measure the cross section of both photosystems in intact, photosynthetically competent cells, without the use of inhibitors or artificial mediators of electron transport. Plots of light-saturation behavior of the respiratory oscillation following pulses at 596 nanometers indicate a mean optical cross section similar to that of PSII at this wavelength, but suggest significant heterogeneity in the cross section of PSI. If this method measures only PSI activity, this result implies that there exist units with different numbers of identical chromophores, or units having populations of chromophores with different absorption spectra.     

Mass spectrometric study of Photosystem II heterogeneity in oxygen and nitrogen production: Effects of magnesium and of phosphorylation of pea thylakoids

Photosystem II (PS II) is capable of the oxidation of both water and hydroxylamine with the production of O₂- and N₂-production, respectively. The resulting changes in the partial pressure of the respective gases can be measured by an appropriate mass spectrometric set-up. Analysis of single turn-over flash saturation curves of O₂- and N₂-production has been performed to determine the relative optical cross sections of the competent PS II units and absolute amounts of their fractions in pea thylakoids. We studied the changes of these parameters upon Mg²⁺-induced transition of thylakoid membrane from unstacked to stacked configuration and upon protein phosphorylation of the stacked samples. The results showed a 2.5-fold increase of effective antenna size of PS II units competent in either O₂- or N₂-production after addition of 10 mM MgCl₂ to cation-depleted thylakoids, which indicates a potential capability of both - and -units to carry out these alternative reactions. However, we observed a significant difference in the amounts of PS II units competent in O₂- or N₂-production, with a ratio of 1:4 in unstacked thylakoids, and reciprocal alterations in stacked ones. This represents an increase by about 20% and a 2-fold decrease of O₂- and N₂-evolving units, respectively, yielding a ratio of 1:1.5, which implies a heterogeneity of PS II with respect to these reactions, the capabilities of - and -units being distinct. The phosphorylation of stacked thylakoids did not essentially influence the antenna size of O₂- and N₂-evolving PS II units but caused opposite and reciprocal changes in their amounts, approximately 30% decrease and increase, respectively, to a ratio of 1:3. The relationship of the structure-function heterogeneity in PS II with implications for current models of photosynthetic regulation mechanisms is discussed.     

Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation.

RNA granules are a macromolecular structure observed in neurons, where they serve as motile units that translocate mRNAs. Isolated RNA granules are highly enriched in Staufen protein and ultrastructurally contain densely packed clusters of ribosomes. With depolarization, many mRNAs, including those involved in plasticity, rapidly shift from the RNA granule fraction to polysomes. Depolarization reorganizes granules and induces a less compact organization of their ribosomes. RNA granules are not translationally competent, as indicated by the failure to incorporate radioactive amino acids and the absence of eIF4E, 4G, and tRNAs. We concluded that RNA granules are a local storage compartment for mRNAs under translational arrest but are poised for release to actively translated pools. Local release of mRNAs and ribosomes from granules may serve as a macromolecular mechanism linking RNA localization to translation and synaptic plasticity.     

Characterization of the nucleation step and folding of a collagen triple-helix peptide

Peptide T1-892 is a triple-helical peptide designed to include two distinct domains: a C-terminal (Gly-Pro-Hyp)(4) sequence, together with an N-terminal 18-residue sequence from the alpha1(I) chain of type I collagen. Folding experiments of T1-892 using CD spectroscopy were carried out at varying concentrations and temperatures, and fitting of kinetic models to the data was used to obtain information about the folding mechanism and to derive rate constants. Proposed models include a heterogeneous population of monomers with respect to cis-trans isomerization and a third-order folding reaction from competent monomer to the triple helix. Fitting results support a nucleation domain composed of all or most of the (Gly-Pro-Hyp)(4) sequence, which must be in trans form before the monomer is competent to initiate triple-helix formation. The folding of competent monomer to a triple helix is best described by an all-or-none third-order reaction. The temperature dependence of the third-order rate constant indicates a negative activation energy and provides information about the thermodynamics of the trimerization step. These CD studies complement NMR studies carried out on the same peptide at high concentrations, illustrating how the rate-limiting folding step is affected by changes in concentration. This sequence preference of repeating Gly-Pro-Hyp units for the initiation of triple-helix formation in peptide T1-892 may be related to features in the triple-helix folding of collagens.     

Number of Deoxyribonucleic Acid Uptake Sites in Competent Cells of Bacillus subtilis

Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of (14)C- or (3)H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell.     

Characterization of messenger RNA populations of Crithidia fasciculata

Cells of Crithidia fasciculata taken from exponentially growing cultures display a relatively high content of poly(A) RNA (roughly 45% of the total) in a nonpolysomal compartment. The proportion of this fraction is significantly higher than that of the free ribosomal units. This RNA is translationally competent and is indistinguishable from polysomal mRNA by various criteria. The significance of these findings is discussed with respect to the protein synthesis capability of the cell and the metabolic relationships between the cytoplasmic compartments of mRNA.     

Action spectra and functional antenna sizes of Photosystems I and II in relation to the thylakoid membrane organization and pigment composition

The functional organization of competent photosynthetic units in developing thylakoids from intermittent-light grown pea as well as in the unstacked, stacked and phosphorylated stacked thylakoids from its mature chloroplasts was characterized by polarographic measurements of action spectra, reaction centre contents and optical cross-sections for PS I-mediated O2 uptake and PS II-mediated O2 evolution. The minimum antenna sizes of 60 and 37 chlorophyll a molecules for PS I and PS II, respectively, were determined in developing thylakoids with a ratio of Chl a/Chl b>50. In mature chloroplasts, the embedded light-harvesting chlorophyll a/b-binding (LHC) protein complexes increased the PS I and PS II effective antenna sizes by 3–6 times depending on the thylakoid membrane organization. In unstacked thylakoids, a randomization of PS I, PS II and LHC II led to the most uniform spectral distribution of light harvesting between the two photosystems but caused the maximal difference of their antenna sizes to be 370 and 100 Chls for the competent PS I and PS II units, respectively. Following the Mg2+-induced stacking of thylakoids, opposite complementary changes of the action spectra, antenna sizes and Chl a/Chl b ratios indicated a redistribution of a LHC II pool of 100 Chl ( a + b) molecules from PS I to PS II. Unlike to the stroma-exposed PS II in unstacked thylakoids, the granal PS II units of 200 Chls demonstrated an additional 2-fold increase of the effective antenna size due to energy transfer within PS II dimers under strong background illumination, which closed >90% of reaction centres. Protein phosphorylation of the stacked thylakoids induced a significant inactivation of the O2-evolving PS II centres but did not cause complementary changes of the action spectra and antenna sizes of the competent PS I and PS II. In this case, light harvesting parameters of the O2-evolving PS II units were nearly unaffected, whereas the obvious relative increase of the PS I activity at 650 nm and its decrease at >700 nm both in the action spectrum and optical cross-section measurements might suggest a substitution of PS I units in the O2-reducing fraction by another distinct fraction of -type which in turn is not the same to PS I units in unstacked thylakoids.     

PSYCHIATRIC UNITS IN GENERAL HOSPITALS-Problems in Development and Efficient Operation

An adequate 25-bed psychiatric unit can be housed in a wing of a general hospital. Even more important than physical facilities are competent personnel, to be headed by a chief psychiatrist and a psychiatric nurse supervisor, for the unit. Incorporating teaching facilities into the unit helps to integrate psychiatry into the other disciplines of medicine in a continuing educational program. Having psychiatric units in general hospitals enables many voluntary patients to be treated in early stages of the disorder, with a high proportion of recoveries. Medicolegal aspects and the lack of adequate coverage of mental disorders by voluntary prepayment health plans are serious problems in the economy of a unit. Improved hospital administration, expanded training programs, educational work by local mental health societies and modification of laws on malpractice and commitment will go far to help solve these problems.     

PSYCHIATRIC UNITS IN GENERAL HOSPITALS—Problems in Development and Efficient Operation

An adequate 25-bed psychiatric unit can be housed in a wing of a general hospital. Even more important than physical facilities are competent personnel, to be headed by a chief psychiatrist and a psychiatric nurse supervisor, for the unit. Incorporating teaching facilities into the unit helps to integrate psychiatry into the other disciplines of medicine in a continuing educational program.Having psychiatric units in general hospitals enables many voluntary patients to be treated in early stages of the disorder, with a high proportion of recoveries.Medicolegal aspects and the lack of adequate coverage of mental disorders by voluntary prepayment health plans are serious problems in the economy of a unit. Improved hospital administration, expanded training programs, educational work by local mental health societies and modification of laws on malpractice and commitment will go far to help solve these problems.     

Defective replication units of hepatitis B virus.

Templates for the synthesis of defective hepatitis B virus RNA pregenomes were constructed. Viral sequences in these constructs were replaced by the neomycin resistance gene. Deletions spanned up to 80% of the genome and did not include the cohesive end region. The size of the defective replication units was reduced up to half of the wild-type unit length. After cotransfection with replication competent wild-type DNA, defective pregenomes became included into the pool of replicating viral nucleic acids. A natural template for a defective pregenome was derived from the integrated state in a hepatocellular carcinoma. Owing to a deletion, this unit was devoid of the hepatitis B virus enhancer.     

Adaptation of sperm whales to open-boat whalers: rapid social learning on a large scale?

Animals can mitigate human threats, but how do they do this, and how fast can they adapt? Hunting sperm whales was a major nineteenth century industry. Analysis of data from digitized logbooks of American whalers in the North Pacific found that the rate at which whalers succeeded in harpooning (‘striking’) sighted whales fell by about 58% over the first few years of exploitation in a region. This decline cannot be explained by the earliest whalers being more competent, as their strike rates outside the North Pacific, where whaling had a longer history, were not elevated. The initial killing of particularly vulnerable individuals would not have produced the observed rapid decline in strike rate. It appears that whales swiftly learned effective defensive behaviour. Sperm whales live in kin-based social units. Our models show that social learning, in which naive social units, when confronted by whalers, learned defensive measures from grouped social units with experience, could lead to the documented rapid decline in strike rate. This rapid, large-scale adoption of new behaviour enlarges our concept of the spatio-temporal dynamics of non-human culture.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Factors affecting genetic transformation of Neisseria gonorrhoeae.

Piliated gonococci were competent in genetic transformation in all stages of growth in minimal and enriched media, but nonpiliated cells were almost totally incompetent. Uptake of deoxyribonucleic acid into a deoxyribonuclease-insensitive state was observed only in competent piliated cells. Competence was not affected by washing of competent cells or treatment of competent cells with proteolytic enzymes. Expression of competence required presence of any of several different monovalent or divalent cations, as well as a utilizable source of energy. Efforts to produce genotypically or phenotypically competent derivatives of nonpiliated cells were unsuccessful. These experiments are consistent with the idea that pili may play a role in the irreversible uptake of transforming deoxyribonucleic acid by the gonococcus, but fail to provide evidence for other types of competence factors.