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H3 receptor antagonists based on a 2-aminoethylbenzofuran skeleton have been discovered, which are potent in vitro at human and rat H3 receptors, with Ki values of 0.1−5.8 nM. Analogues were discovered with potent (0.01−1 mg/kg) cognition and attention enhancing properties in animal models. One compound in particular, 4-(2-[2-(2(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl)benzonitrile (ABT-239), combined potent and selective H3 receptor antagonism and excellent pharmacokinetic and metabolic properties across species, with full efficacy in two behavioral models:  a five-trial inhibitory avoidance acquisition model in rat pups at 0.1 mg/kg and a social recognition memory model in adult rats at 0.01 mg/kg. Furthermore, this compound did not stimulate locomotor activity and showed high selectivity for the induction of behavioral efficacy versus central nervous system based side effects. The potency and selectivity of this compound and of analogues from this class support the potential of H3 receptor antagonists for the treatment of cognitive dysfunction.

To compare the use of surgical treatment for epilepsy among different ethnic and racial groups with surgically remediable temporal lobe epilepsy (TLE).

A nanostructured, porous material was prepared by heterocoagulation of negatively charged montmorillonite with positively charged synthetic spherical hematite particles. The process of heterocoagulation of such particles was monitored by turbidimetric titrations over the pH range 2.5−7.5. On the basis of the results of turbidimetric measurements, a series of solid materials were prepared for further characterization using ESEM, BET, XRD, and FTIR techniques. Environmental scanning electron microscopy detected isolated hematite particles or small hematite aggregates on montmorillonite surfaces (mass ratios 8:1 and 4:1). At a mass ratio of 1:1, exfoliated montmorillonite layers, covering the hematite particles as semi-transparent blankets were seen. A low mass ratio led to compact hematite particle aggregates covering the montmorillonite surfaces. Nitrogen-gas-adsorption isotherms revealed the sorption properties to be gradually dependent upon mass ratios. Pore volume distributions showed that mesopores with diameter of about 10−20 nm were produced in the heterocoagulates with mass ratios of 4:1, 1:1, and 1:8. The sample prepared with a 4:1 mass ratio showed the greatest BET surface area, which decreased slightly upon sample calcination at 500 °C. X-ray diffraction measurements were used to investigate layer stacking, by comparing the integral intensities of d001 reflection. For this purpose, samples with 4:1 mass ratios, prepared both by heterocoagulation and mechanical grinding, were used. It was found that heterocoagulation effectively diminished the stacking of the layers to about 85%; hence, a significant amount of fundamental, 1 nm thick montmorillonite layers was achieved in this sample.

Recombinant activated factor VII (rFVIIa, Novo Nordisk A/S, Bagsvaerd, Denmark) is increasingly being used to secure hemostasis in difficult clinical situations. The role of rFVIIa in the treatment of patients undergoing open‐heart surgery for valvular heart disease was evaluated in an open pilot study. The study objectives were to evaluate blood loss and hemostatic effect following the administration of rFVIIa, to evaluate the safety of the treatment, and to determine its effect on laboratory parameters. To date, we have treated five patients (one child aged 2.5 years and four adults) using various surgical procedures, including arterial switch, closure of atrial septal defect and DeVega's procedure (mitral valve replacement with tricuspid valve repair). Four patients received rFVIIa intraoperatively while the fifth received it postoperatively. Satisfactory hemostasis was achieved with a single dose (30 μg/kg) of rFVIIa. Four hours after treatment with rFVIIa, the mean blood loss was 262.5 mL for adults (220–334 mL) and 85 mL for the child. No significant adverse events were seen in relation to rFVIIa therapy. The laboratory parameters indicated a mean 18.5‐fold increase (range 3.7–42) in FVII levels at 30 min post‐injection and a mean reduction of 12 s (range 3–39 s) in prothrombin time.

It is generally assumed that men with congenital bilateral absence of the vas deferens (CBAVD) have azoospermia because of obstruction and that sperm production is normal. This study examines spermatogenesis in men with CBAVD to assess the validity of this assumption. We identified all men with CBAVD who had undergone either a diagnostic or therapeutic fertility procedure. Procedures included diagnostic biopsy, testis fine needle aspiration (FNA) mapping, microscopic epididymal sperm aspiration (MESA), and testis sperm extraction (TESE). Among 33 CBAVD men, 18 underwent testis biopsy, 27 had MESA/TESE, and 10 had FNA mapping. On evaluation of these procedures, normal spermatogenesis was present in 29 men. Four men (12%) demonstrated impaired spermatogenesis. One patient had FNA testis cytology consistent with late maturation arrest, another demonstrated hypospermatogenesis on biopsy and low sperm yield by MESA, and two patients had pure Sertoli cell only histology on biopsy. Aetiologies for impaired spermatogenesis included varicocele and underlying genetic abnormalities. Although patients with CBAVD are assumed to have normal spermatogenesis and infertility due simply to obstruction, the potential for concomitant defects in sperm production exists. A clinical suspicion of testis failure should prompt further diagnostic evaluation of spermatogenesis prior to sperm retrieval. In addition, genetic counselling should be offered and testing for genetic lesions, including cystic fibrosis gene mutations and/or variants, Y chromosome microdeletions, and karyotype abnormalities, should be considered.

The sorption behavior of Cs+, and Ba2+on natural clay was investigated using ToF-SIMS, XPS, and XRD. The natural clay was composed mainly of chlorite and illite in addition to quartz and calcite. Depth profiling up to 70 Å was performed at 10 Å steps utilizing ToF-SIMS to study the amount of sorbed Cs+and Ba2+as a function of depth in the clay matrix. The results suggest that Cs+and Ba2+ ions were sorbed primarily by ion exchange coupled with hydrolytic sorption. According to ToF-SIMS and XPS results, the total sorbed amount of Ba2+was larger than that of Cs+. Quantitative determination of the primary cations within the analyzed clay before and after sorption indicated that for Ba2+sorption, Ca2+, Mg2+and for Cs+sorption Ca2+, K+ were the major exchanging ions. The XRD spectra of Ba-sorbed clay contained new peaks that were identified as BaCO3.

A study is reported of the influence of organic acids (particularly acetic acid) on the development of lead patinas. Raman spectroscopy has been used to determine the chemical composition of patinas, while mass loss on descaling and scanning electron microscopy have followed their physical development. It was found that the mechanism of the patination process initially involved the formation of an oxide film which rapidly converted to a basic lead carbonate via normal and basic lead acetate intermediates. The high solubility of the acetate intermediaries influenced the morphology of the patina and increased the extent of its formation.

A study of the factors influencing lead patination is reported. Raman spectroscopy has been used to determine the composition of patinas, while weight gain measurements and scanning electron microscopy have followed their physical development. The mechanism of the pati nation process follows a route during which the initially formed oxide film converts to a basic lead carbonate (hydrocerussite, 2PbCO3. Pb(OH)2), which, under the influence of sulphur dioxide, ultimately forms lead sulphate, via intermediate tetrabasic lead sulphate and lead sulphite phases. Levels of atmospheric pollution were observed to play an important role in the rate of formation of the stable end product.

The bacteriophage T4 GroES homologue, gp31, in conjunction with the Escherichia coli chaperonin GroEL, is both necessary and sufficient to fold the T4 major capsid protein, gp23, to a state competent for capsid assembly as shown by in vivo expression studies. GroES is unable to function in this role as a productive co-chaperonin. The sequencing and characterization of mutations within gp23 that confer GroEL and gp31 chaperonin-independent folding of the mutant protein suggest that the chaperonin requirements are due to specific sequence determinants or structures in critical regions of gp23 that behave in an additive fashion to confer a chaperonin bypass phenotype. Conservative amino acid substitutions in these critical regions enable gp23 to fold in a GroEL-gp31 chaperonin-independent mode, albeit less efficiently than wild type, both in vivo and in vitro. Although the presence of functional GroEL-gp31 enhances folding of the mutated gp23 in vivo, GroEL-GroES has no such effect. Site-directed mutagenesis experiments suggest that a translational pausing mechanism is not responsible for the bypass mutant phenotype. Polyhead reassembly experiments are also consistent with direct, post-translational effects of the bypass mutations on polypeptide folding. Given our finding that gp31 is not required for the binding of the major capsid protein to GroEL and that active GroES is incapable of folding the gp23 polypeptide chain to native conformation, our results suggest co-chaperonin specificity in the folding of certain substrates.

Methylene-bis-propionate (MBP) was compared to a 15% formaldehyde solution (FOR) and propionic acid (PA) as a preservative for high-moisture corn (HMC). In a field trial, MBP, FOR and PA at concentrations of .78%, 1.00% and .80% (wt preservative/wt corn), respectively, were applied to 70.4 M3(2,000 bu) lots of freshly harvested yellow dent corn (20 to 26%) moisture). Microbiological changes were evaluated throughout 240 days of storage in concrete silos, and the performance of 25 feedlot heifers was determined on corn treated with each of the preservatives. MBP exhibited superior antifungal and antibacterial properties throughout storage, whereas FOR controlled fungi and bacteria less than 20 days. PA controlled microbial activity during most of storage but was less effective than MBP. Average daily gain (kg) and feed-to-gain ratio for the respective (MBP, FOR and PA) rations were: 1.00 and 6.78, .97 and 6.85 and 1.10 and 6.04. Laboratory experiments were conducted to determine the fat of MBP on HMC. MBP broke down to PA and formaldehyde within 9 hr after contacting the wet grain. This breakdown appears to be a hydrolytic process which is catalyzed by a heat-sensitive substance. Minimum levels of MBP preserved high-moisture corn in laboratory tests longer than mixtures of PA and FOR.

Serum samples and body weights were taken at regular intervals for six to seven months from piglets, born to foot-and-mouth disease (fmd) vaccinated or unvaccinated sows, which had been vaccinated at one, two, four or eight weeks old. Young pigs, devoid of maternally derived antibodies (mda), were capable of responding to fmdvaccination at one week old, with no deleterious effects on their growth rate. However, their immunity to experimental infection at six to seven months old was poor (33·3 per cent). Vaccination of four or eight-week-old piglets, born to unvaccinated sows, protected 87·5 per cent from a similar challenge. In piglets, born to fmdvaccinated sows, the mdahad a suppressive effect on the early vaccination response. This suppression, which was affected by the titre of mdapresent in the piglets at •he time of vaccination, was complete in one-, two-and four-week-old piglets and partial in eight-week-old piglets. Furthermore, none of these piglets were immune to experimental infection at six to seven months old.

Groups of 68 and 66 cattle aged 12 and 24 months respectively were each subdivided into 16 groups and inoculated with foot-and-mouth disease vaccines containing O1Campos, A24Cruzeiro and C3Pando virus strains. The 140S antigen mass of the O1and A24valencies was varied while that of C3was held constant. Multifactorial comparisons between the 21 day serum neutralising antibody titres showed that over most of the range there was a linear log dose response relationship. Doubling the antigen dose increased the serum antibody litres against both A24Cruzeiro and O1Campos by approximately 0·15 log10. The A24antigen was about 30 times more immunogenic than the O1with C3intermediate between the two. At high antigen doses the responses flattened but the level at which this occurred depended on the immunogen administered. No difference could be demonstrated between the responses of 12- and 24-month-old cattle and there was no evidence of competitive inhibition or enhancement between the virus strains included in the vaccines.

The multigene family which codes for the mouse major urinary proteins consists of about 35 genes. Most of these are members of two distinct groups, group 1 and group 2. The group 1 and group 2 genes are organized in head-to-head pairs within 12 to 15 remarkably uniform chromosomal units or domains about 45 kilobase pairs (kb) in size. The 45-kb units are located on chromosome 4, and many of them are adjacent to each other. We propose that the 45-kb unit is a unit both of organization and of evolutionary change. In this study the homologies within the unit were observed by examining, in an electron microscope, heteroduplex and foldback structures made from cloned major urinary protein genes. These show that the 45-kb unit is a gigantic imperfect palindrome. Each arm of the palindrome contains two regions of inverted symmetry of 9.5 and 4.5 kb separated by a 3-kb nonsymmetrical region. We argue that the nonsymmetrical regions arose by a series of deletion events in the two arms of the palindrome. The center of the 45-kb unit is an 8-kb sequence without inverted symmetry flanked by the 9.5-kb regions, which contain the 4-kb genes and their immediate 5' and 3' flanking regions. The junction between adjacent 45-kb units is a 2- to 4-kb sequence without inverted symmetry flanked by the 4.5-kb regions. Some of the 45-kb units are arranged as direct tandem repeats. Others appear to be in inverted orientation with respect to a neighboring unit. Cloned major urinary protein genes show few incidences of the repetitive elements B1, B2, R, and MIF. Two elements, a B1 and an R, may be a constant feature of the 45-kb units. If so, in those cases in which the units are in tandem array, both of these elements will occur with a 45-kb periodicity. A comparison of corresponding parts of different 45-kb units shows that they differ because of a number of deletion or insertion events, particularly in the regions 3' to the genes.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible and nonselective protein-modifying reagent, has been used extensively in studies involving inactivation of receptors. Here, we present N-(p-isothiocyanatophenethyl)spiperone (NIPS), a novel and highly selective irreversible inactivator of D2 but not D1 receptors. In in vitro studies, NIPS exhibited an apparent Ki of 10 nM for [3H]methylspiperone binding to D2 receptors in rat striatum. Preincubation of the striatal membranes with NIPS followed by extensive washing resulted in up to an 80% reduction of the D2 receptor maximum binding (Bmax). Coincubation with the D2 receptor antagonist domperidone could protect against this reduction. NIPS was additionally shown to irreversibly inactivate D2 receptor binding activity in cultured cells expressing the D2 receptor protein. In in vivo administration studies, using [3H]SCH 23390 and [3H]spiperone to assay D1 and D2 receptors in vitro, 24 hr after injection (s.c.) with 5 to 40 mg/kg of NIPS D2 receptor, Bmax was decreased by 58 to 76%, without a change in D2 receptor affinity. In contrast, there was no effect on D1 receptor Bmax or affinity. There was also a small (24%) reduction in frontal cortex 5-hydroxytryptamine2 receptors by 20 mg/kg of NIPS. However, there was no effect on alpha-1 or alpha-2 adrenergic receptors in the frontal cortex, or on muscarinic cholinergic or 5-hydroxytryptamine1A receptors in the hippocampus. After single doses of either 20 mg/kg of NIPS or 10 mg/kg of EEDQ, the D2 receptor recovery rate was much slower after NIPS (half-time of receptor recovery = 170 hr) than after EEDQ (half-time of receptor recovery = 76.7 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homogeneity from an extract of late infected Escherichia coli. Both DNA-dependent ATPase and endonuclease activities co-chromatograph, co-sediment, and have been renatured from a single 43-kilodalton protein eluted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that both activities are exerted by one multifunctional protein. Duplex, single-stranded, and supercoiled DNAs are all effective activators of the high specific activity ATPase which produces ADP and inorganic PO4. The enzyme displays a broad specificity towards the nucleoside and deoxynucleoside triphosphates, and the ATPase activity is strongly inhibited by DNA-intercalating compounds. The endonuclease appears to be most active on supercoiled DNA, producing double-stranded breaks in duplex DNA, and does not require nucleoside triphosphates. An antiserum against the purified enzyme immunoprecipitated it, inhibited its ATPase activity, and also precipitated from extracts a T4-induced protein of Mr = 43,000. This antigen was not found in uninfected E. coli, or following a gene 55am mutant (late protein synthesis defective) infection, and was not detected following infection with T4 amber mutants of any early capsid protein gene which blocks T4 head protein cleavage in vivo. In a pulse-chase experiment, the radioactive antigen was not found following a pulse of radioactive amino acids, but appeared after a chase with excess nonradioactive amino acids. The enzyme-related antigen is apparently produced by cleavage of a precursor by the T4 head assembly proteinase which processes a number of prohead proteins. These processing reactions are dependent in vivo upon assembly of the prohead and are required for its maturation. The evidence suggests that this enzyme functions in head assembly and DNA packaging, and originates as the cleavage product of a prohead precursor protein.

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.

T4 DNA structural requirements for encapsidation in vivo were investigated, using thin-section electron microscopy to quantitate the kinetics and yields of head intermediates after synchronous DNA packaging into accumulated processed proheads. UV irradiation (254 nm) of T4-infected bacteria just before initiation of encapsidation resulted in a reduction in the rate of DNA packaged measured by electron microscopy and in the yield of viable phage progeny. In UV-irradiated infections with excision-deficient mutants (denV-), the extent of packaging decline was proportional to the UV dose and phage yields were lower than expected based on the packaging levels observed by microscopy. Rescue analysis of progeny from such infections revealed elevated levels of nonviable virions. Pyrimidine dimers were encapsidated in denV- infections, but in excision-competent infections (denV+) dimers were not packaged. A UV-independent, 15 to 20% packaging arrest was also observed when denV endonuclease was inactive during encapsidation, indicating a denV requirement to achieve normal T4 packaging levels. Pyrimidine dimers apparently represent or induce transient blockage of DNA encapsidation or both, causing a decline in the rate. This is in contrast to other DNA structural blocks to packaging induced by mutations in T4 genes 30 and 49, which appear to arrest the process.

Y-79 human retinoblastoma cells can be induced to express significant quantities of functional D2 dopamine receptors after attachment and differentiation with sodium butyrate. In membranes prepared from differentiated Y-79 cells, the D2 dopaminergic antagonist [3H] methylspiperone exhibits a KD of 77 pm and a Bmax of 60 fmol/mg of protein, whereas the antagonist [125I]iodosulpride reveals a KD of 0.77 nM and a Bmax of 40 fmol/mg of protein. Dopamine also induces a pharmacologically specific, pertussis toxin-sensitive, dose-dependent inhibition of forskolin-stimulated adenylyl cyclase activity, with an EC50 of 2 microM and a maximal response at 100 microM (approximately 50% enzyme inhibition). Pretreatment of the cells with dopamine results in a diminution in the subsequent ability of dopamine to inhibit adenylyl cyclase activity. This effect is time dependent, reaching maximal desensitization after approximately 24 hr. The dopamine dose-response curve for inducing desensitization exhibits an EC50 of approximately 2-3 microM and a maximal response at approximately 0.1-1 mM, similar to that for inhibiting adenylyl cyclase activity. After maximal desensitization, the EC50 for dopamine-induced inhibition of adenylyl cyclase activity is increased greater than 20 fold (lower affinity) and the maximum inhibition is decreased to approximately 15%, representing an approximately 70% desensitization. The agonist-induced desensitization is pharmacologically specific, inasmuch as preincubation of the cells with the dopaminergic agonists epinine and (+-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene or the D2-selective agonist N-0434 also results in desensitization of dopamine-induced inhibition of enzyme activity, whereas preincubation with the D1-selective agonist SKF-38393 or with the nondopaminergic agonists isoproterenol and serotonin results in little or no desensitization. Preincubation of the cells with dopamine also promotes a time-dependent increase (approximately 3-fold) in the KD for [3H]methylspiperone, with no change in its Bmax. In contrast, after dopamine preincubation, the KD for [125I]iodosulpride is unchanged, whereas its Bmax is reduced by approximately 50% upon maximum desensitization. In addition, agonist pretreatment promotes a functional uncoupling of the D2 receptor, as suggested by a loss of high affinity agonist binding observed in radioligand competition binding assays after desensitization. Upon removal of agonist, the cellular D2 receptor binding activity and functional response recover to control levels within a 24-hr period. These results suggest that prolonged exposure of cells to dopaminergic agonists initiates a desensitization process involving a functional uncoupling of the D2 dopamine receptor as well as a loss of its ligand binding activity.

Phage T4 terminase is an enzyme that binds to the portal protein of proheads and cuts and packages concatemeric DNA. The T4 terminase is composed of two subunits, gene products (gp) 16 and 17. The role of the small subunit, gp16, in T4 DNA packaging is not well characterized. We developed a new purification procedure to obtain large quantities of purified gp16 from an overexpression vector. The pure protein is found in two molecular weight forms, due to specific C-terminal truncation, displays in vitro packaging activity, and binds but does not hydrolyze ATP. gp16 forms specific oligomers, rings, and side-by-side double rings, as judged by native polyacrylamide gel electrophoresis and scanning transmission electron microscopy measurements. The single ring contains about eight monomers, and the rings have a diameter of about 8 nm with a central hole of about 2 nm. A DNA-binding helix-turn-helix motif close to the N terminus of gp16 is predicted. The oligomers do not bind to DNA, but following denaturation and renaturation in the presence of DNA, binding can be demonstrated by gel shift and filter binding assays. gp16 binds to double-stranded DNA but not single-stranded DNA, and appears to bind preferentially to a gene 16-containing DNA sequence.

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction.

We analyzed by restriction enzyme digestion the end structure of T4 phage DNA by comparing mature, concatemeric, first-packaged, and incompletely packaged DNAs. The structure of mature DNA was also studied using 3' end labeling with terminal transferase. Our data support the hypothesis that T4 DNA packaging is not initiated at specific packaging initiation sequences on the concatemeric precursor (cos or pac site mechanisms) but by a different packaging mechanism.

A survey of DNA packaging in vivo and in vitro during infections caused by T4 DNA-delay and DNA-arrest amber mutants revealed a common DNA packaging-deficient phenotype. Electron microscopy revealed high proportions of proheads partially filled with DNA in vivo, indicating normal initiation but incomplete encapsidation. In contrast, exogenous mature T4 DNA was packaged in vitro by several early-gene mutant extracts. Detailed analysis of gene ts39 mutants (subunit of topoisomerase II) showed that in vivo packaging is defective, yet expression of late proteins appeared normal and the concatemeric DNA was not abnormally short or nicked. Although g39 amber mutant extracts packaged DNA in vitro, two of three ts39 mutant extracts prevented encapsidation of the exogenous DNA. The temperature-sensitive (ts) gp39 in a mutant topoisomerase II complex may have interfered with packaging in vivo and in vitro by interacting with DNA in an anomalous fashion, rendering it unfit for encapsidation. These results support the hypothesis that T4 DNA packaging is sensitive to DNA structure and discriminates against encapsidation of some types of defective DNA.

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.

Ion etching of bacteriophage T4 erodes virus components progressively from the outside to the inside while preserving the overall structure. The terminal portion of the T4 DNA molecule packaged can be specifically radiolabeled and was found to be eroded more rapidly than the remainder of the DNA. This strongly suggests that the first DNA to enter the prohead is condensed in the center of the capsid and is therefore shielded from the ion beam by the surrounding last packaged DNA. The results support a "spiral-fold" model for the arrangement of DNA within the icosahedral bacteriophage head. According to this model, phage T4 DNA strands run parallel to the long axis of the phage, with sharp (180 degrees) bends at the top and bottom of the capsid. The folds themselves are arranged radially about the long axis of the head in spirally organized shells.

Demonstrates how the Santa Fe home of Shane and Jordan Cronenweth reflects the beauty of the surrounding landscape. Use of color, shape and texture; Use of furnishings made from the region's natural resources; Use of oversized furniture; Photos of various rooms.

Electron micrographs of sections cut through tobacco leaves infected with tobacco mosaic indicate that the virus is often present in fibrous masses associated with chloroplasts which are severely affected. Virus can clearly be seen in the cytoplasm of the diseased cells sometimes as single filaments. The nuclei seem unaltered by the infective process and have appeared free of virus. The great length of these filaments, much in excess of 2800A, indicates an end-to-end arrangement of the rods in situ.

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 106Staphylococcus aureuscells per ml. When similar milks were inoculated with 104S. aureuscells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 106S. aureuscells per ml and incubated at 35 C. Generally, a concentration of 5 × 107S. aureuscells per ml of milk was reached before enterotoxin A was detected.