The effect of light fluence rate in photodynamic therapy of normal rat brain.

This paper reports the effect of incident light fluence rate on the depth to which necrotic lesions are produced by photodynamic therapy (PDT) in the brains of normal Fisher rats. The rats were injected intraperitoneally with Photofrin (12.5 mg kg-1) 48 h prior to PDT with a fixed incident fluence of 35 J cm-2. The treatment was performed at 10, 50, 100, and 200 mW cm-2 and also in a periodic manner (30 s "on" at 100 mW cm-2, 30 s "off"). The depth to which necrosis occurred was determined 24 h after treatment by microscopic examination of tissue sections. No differences were found in the depth to which necrosis was produced by any of the five irradiation schedules. This finding is discussed in the context of other published dose-rate experiments.     

Production and cytogenetic analysis of BC1, BC2, and BC3 progenies of an intergeneric hybrid between Triticum aestivum (L.) Thell. and tetraploid Agropyron cristatum (L.) Gaertn.

Summary Intergeneric hybrids between Triticum aestivum cv Chinese Spring and Agropyron cristatum 4x (2n= 5x=35, ABDPP genomes) with a high level of homoeologous meiotic pairing between the wheat chromosomes were backcrossed 3 times to wheat. Pollination of the F₁ hybrid with Chinese Spring resulted in 22 BC₁ seeds with an average seed set of 1.52%. Five BC₁ plants with 39–41 chromosomes were raised using embryo rescue techniques. Chromosome pairing in the BC₁ was characterized by a high frequency of multivalent associations, but in spite of this there was no evidence of homoeologous pairing between chromosomes of wheat and those of Agropyron. All of the plants were self sterile. The embryo rescue technique was again essential to produce 39 BC₂ plants with chromosome numbers ranging from 37 to 67. The phenomenon of meiotic non-reduction was also observed in the BC₃ progenies. In this generation male and female fertility greatly increased, and meiotic pairing was fairly regular. Some monosomic (2n=43) and double monosomic (2n=44) lines were produced. Analysis of these progenies should permit the extraction of the seven possible wheat-Agropyron disomic addition lines including those with the added chromosomes carrying the genes involved in meiotic non-reduction and in suppression of Ph activity.     

A Brassica campestris-alboglabra addition line and its use for gene mapping,intergenomic gene transfer and generation of trisomics

Summary Brassica campestris-alboglabra monosomic addition lines were developed from a trigenomic Brassica hybrid (2 n=3 x=29, AAC) obtained by backcrossing a resynthesized B. napus (2 n=4 x=38, AACC) line to its parental B. campestris (2 n=2 x=20, AA) line. One addition line was characterized genetically with three loci specific for the alien chromosome and cytologically by meiotic analysis. The following results were obtained. (1) The same chromosome in the B. alboglabra (2 n= 2 x=18, CC) genome carried the three loci, E _c, W _c and Lap-1 C ^c, which control the biosynthesis of erucic acid, white flower colour and the faster migrating band of leucine aminopeptidase, respectively. The linear order and possible positions of the three loci were inferred. The meiotic behaviour of the alien chromosome was documented and its transmission frequency was assessed. (2) Intergenomic recombination frequently occurred in the monosomic addition line, resulting in the introgression of one or two loci from the alien chromosome into the B. campestris genome. (3) B. campestris trisomics were found in the progeny of the monosomic addition line. (4) The removal of the other eight C-genome chromosomes from the trigenomic Brassica hybrid led to a dramatic increase in the erucic acid content of the monosomic addition line. (5) No offspring of the trigenomic Brassica hybrid showed evidence of intergenomic recombination and introgression of the W _c locus into the B. campestris genome. It is questioned whether such a difference might be due to a possible regulating mechanism for homoeologous chromosome pairing.     

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands.

Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.     

Production of the Mycotoxin Fumonisin B(1) by Alternaria alternata f. sp. lycopersici

The mycotoxin fumonisin B(1), originally described as being produced by Fusarium moniliforme, was detected in liquid cultures of Alternaria alternata f. sp. lycopersici, a host-specific pathogen of tomato plants. The metabolite was detected by high-pressure liquid chromatography and mass spectrometry. Its identity was confirmed by fast atom bombardment and ion spray mass spectrometry, as well as parent-daughter tandem mass spectrometry. In three separate experiments, the concentrations found ranged between 5 and 140 ppm (mug/ml).     

High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone.

Fermentation studies were performed on an Escherichia coli culture that carries a recombinant plasmid composed of an ampicillin-resistant gene, a temperature-regulated pL promoter, and a porcine pituitary cDNA sequence coding for growth hormone. The objective was to achieve high cell density while maintaining the specific expression level of recombinant porcine growth hormone (r-pGH) observed in shake flasks. At a specific expression level of 20% of total cell protein, the cell density of a glucose-limited fed-batch process reached 38 units of OD600 in 14 h, compared to flask cultivation, which resulted in only 1.4 units of OD600 in the same period. The observed critical fermentation conditions for maximal expression included (1) limiting glucose concentration below 1 g l-1 throughout the fed-batch growth and induction phases, (2) keeping postinduction temperature at 42 degrees C for 5-7 h, and (3) maintaining a postinduction growth rate around 0.17-0.21 h-1.     

Facile preparation of optically pure [α-2H]-α-amino acids

Summary Racemic [-²H]--amino acids were prepared by heating the corresponding amino acids (Phe, nor-Leu and Dopa) with 0.05 equivalents of benzaldehyde in deuterated-acetic acid. Based on¹H-nmr measurement, the isotopic purities of these racemized [-²H]--amino acids were found to be higher than 99.5%. Methylation of these isotope-labelled amino acids was achieved in methanol/thionyl chloride without affecting isotopic purity. Optically pure [-²H]--amino acids were obtained in high yield with high enantiomeric excess via alcalase catalysed resolution.     

The effects of large blood vessels on temperature distributions during simulated hyperthermia.

Several three-dimensional vascular models have been developed to study the effects of adding equations for large blood vessels to the traditional bioheat transfer equation of Pennes when simulating tissue temperature distributions. These vascular models include "transiting" vessels, "supplying" arteries, and "draining" veins, for all of which the mean temperature of the blood in the vessels is calculated along their lengths. For the supplying arteries this spatially variable temperature is then used as the arterial temperature in the bioheat transfer equation. The different vascular models produce significantly different locations for both the maximum tumor and the maximum normal tissue temperatures for a given power deposition pattern. However, all of the vascular models predict essentially the same cold regions in the same locations in tumors: one set at the tumors' corners and another around the inlets of the large blood vessels to the tumor. Several different power deposition patterns have been simulated in an attempt to eliminate these cold regions; uniform power in the tumor, annular power in the tumor, preheating of the blood in the vessels while they are traversing the normal tissue, and an "optimal" power pattern which combines the best features of the above approaches. Although the calculations indicate that optimal power deposition patterns (which improve the temperature distributions) exist for all of the vascular models, none of the heating patterns studied eliminated all of the cold regions. Vasodilation in the normal tissue is also simulated to see its effects on the temperature fields.(ABSTRACT TRUNCATED AT 250 WORDS)     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation