The rtn gene of Proteus vulgaris is actually from Escherichia coli.

The rtn gene, identified as coming from Proteus vulgaris ATCC 13315, is present in Escherichia coli K-12, and over a 440-bp region of rtn is identical to the published Proteus sequence, with the exception of a single G insertion. It was not possible to verify the presence of rtn in P. vulgaris.     

Structural alterations in synaptosomal membrane-associated proteins and lipids by transient middle cerebral artery occlusion in the cat

We have previously reported that ischemia reperfusion injury results from free radical generation following transient global ischemia, and that this radical induced damage is evident in the synaptosomal membrane of the gerbil. [Hall et al, (1995) Neuroscience 64: 81–89] In the present study we have extended these observations to transient focal ischemia in the cat. We prepared synaptosomal membranes from frontal, parietal-temporal, and occipital regions of the cat cerebral cortex with reperfusion times of 1 and 3 hours following 1 hour right middle cerebral artery occlusion. The membranes were selectively labeled with protein and lipid specific paramagnetic spin labels and analyzed using electron paramagnetic resonance spectrometry. There were significant motional changes of both the protein and lipid specific spin labels in the parietal-temporal and occipital regions with 1 hour reperfusion; but, both parameters returned to control values by 3 hours reperfusion. No significant changes were observed in the normally perfused frontal pole at either reperfusion time. These results support the argument that free radicals play a critical role in cell damage at early reperfusion times following ischemia.     

Characterization of translation products of the polyadenylated RNA of free and membrane-bound polyribosomes of rat forebrain.

Poly(A)+ RNA (polyadenylated RNA) isolated from membrane-bound and free polyribosomes was translated in reticulocyte lysates, and the products were analysed by two-dimensional gel electrophoresis. Several translation products were specific to membrane-bound polyribosomal mRNA, including polypeptides of 47kDa, 35kDa and 21 kDa, whereas others (e.g. of 37 kDa, 17 kDa and 14 kDa) were specific to free polyribosomal mRNA. Although many products were common to both mRNA species, cross-contamination could be ruled out on the basis of the presence of these and other specific products. The common products included a 68 kDa microtubule-associated protein, tubulin, actin, the brain form of creatine kinase, neuron-specific enolase and protein 14-3-3 and calmodulin, all of which were identified on the basis of two-dimensional gel and peptide analyses. The 35 kDa protein product of membrane-specific mRNA was co-translationally processed in vitro by microsomal membranes, resulting in its cleavage to 33 kDa (and partial glycosylation). The 33 kDa processed protein (but not the 35 kDa precursor) was integrated into both dog pancreas and rat brain microsomal membranes. The occurrence of the enzymes and calmodulin as products of membrane-bound polyribosomal mRNA is discussed in the light of their presence on rat brain synaptic plasma membranes [Lim, Hall, Leung, Mahadevan & Whatley (1983) J. Neurochem. 41, 1177-1182] and their existence in a specific component of axonal flow. It is suggested that some of these translation products of the rough endoplasmic reticulum may represent proteins destined for the plasma membrane. However, the identity and location of the 35 kDa membrane-specific product (or its processed form) still remain unestablished.     

Positive-negative, chemical-ionization, direct exposure mass spectrometry of underivatized prostaglandins

Nine underivatized prostaglandins were examined using direct exposure, ammonia, chemical-ionization, pulsed positive-negative ion mass spectrometry. The positive ion spectra were characterized by (M+18)+ ion adducts. The negative ion spectra were characterized by ions which depended upon the functionality present in the cyclopentane ring system (acetal for TXB2). The E and D series prostaglandins gave (M-18)- as the major negative ion, while the F series and TXB2 were characterized by negative ions corresponding to (M-1)-, and PGA2 by the parent (M)- ion. Prostaglandin 6-keto-PGF1 alpha was anomalous in this respect showing apparent dehydration, interpreted as an overall (M-18+1)+ and (M-18-1)- in the positive and negative ion spectra, respectively. All major ion types were shown to give essentially a linear response with respect to concentration in the 10-1000 ng range. Although these initial studies were conducted under ideal conditions, it would appear that direct chemical ionization techniques show promise for providing direct structural information on prostaglandins without the need for prior chemical derivatization.     

Application of the Indirect Immunoperoxidase Stain Technique to the Flagella of Azospirillum brasilense

An indirect immunoperoxidase stain was used to demonstrate by electron microscopy that an antigenic difference exists between the polar flagellum and the lateral flagella of Azospirillum brasilense ATCC 29145.     

Number of mutations required to evolve a new lactase function in Escherichia coli.

The frequency of mutation of the ebgAo allele to ebgA+ was compared with the frequency of mutation of strA+ to strA-. The observation that both spontaneous and ethyl methane sulfonate-induced mutations to ebgA+ occurred more frequently than mutations to strA- suggests that ebgA+ mutants arise as the result of single-point mutations.     

Some improvements in two-dimensional gel electrophoresis of proteins. Protein mapping of eukaryotic tissue extracts.

In the course of adapting O'Farrell's (1975, J. Biol. Chem.250, 4007–4021) two-dimensional separation technique for proteins to eukaryotic material, we have made some modifications. During sample preparation, sodium dodecyl sulfate (SDS) can be included, with a resulting enhancement in reproducibility of gel patterns. However, heating in the presence of SDS leads to artifactual spots in the gels, probably as a result of protein charge modifications. Ultracentrifugation reduces the clogging at the top of the isoelectric focussing gel. For electrophoresis, some modifications of apparatus and technique are suggested. For the analysis of gels, a simple high-efficiency method for the counting of radioactivity in spots from dried gel slabs is described. In addition, an inexpensive microdensitometer option is described for the analysis of the autoradiographs. Patterns of proteins obtained from superior cervical sympathetic ganglia of rats and from other eukaryotic tissues are illustrated. Finally, a few of the proteins commonly found in mammalian tissue are identified on the gels.     

A model of leaf photosynthesis and respiration for predicting carbon dioxide assimilation in different environments

Summary A model of leaf photosynthesis and repiration was developed which adequately predicted carbon dioxide assimilation responses by a C ₃ species, Atriplex patula, to light, [CO₂], [O₂] and temperature in controlled environments. Methods were developed for estimating input parameters using laboratory, controlled environment and field data.     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.