Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 10⁴. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85C for 10 min did not reduce the viable count by a factor of 10⁴. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum . These findings are relevant to the safety of minimally processed (e.g. sous-vide ) foods.     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

Land use,rangeland degradation and ecological changes in the southern Kalahari,Botswana

Dual‐scale analyses assessing farm‐scale patterns of ecological change and landscape‐scale patterns of change in vegetation cover and animal distribution are presented from ecological transect studies away from waterpoints, regional remotely sensed analysis of vegetation cover and animal numbers across the southern Kalahari, Botswana. Bush encroachment is prevalent in semi‐arid sites where Acacia mellifera Benth. is widespread in communal areas and private ranches, showing that land tenure changes over the last 40 years have not avoided rangeland degradation. Herbaceous cover is dominated in intensively grazed areas by the annual grass Schmidtia kalahariensis Stent and in moderately grazed areas by the perennial grass Eragrostis lehmanniana Nees. Nutritious perennial grass species including Eragrostis pallens Hack. Ex Schinz remain prevalent in Wildlife Management Areas. Other ecological changes include the invasion of the exotic Prosopis glandulosa Torr. and dense stands of Rhigozum trichotomum Kuntze. in the arid southwest. Regional patterns of wildlife species show that the expansion of cattleposts and fenced ranches has led to large areas of low wildlife conservation value even in areas where cattle production is not practiced. Findings show the need for integrated landscape‐scale planning of land use if the ecological value and biodiversity of the southern Kalahari is to be retained.     

The synthesis of a DOPA analogue, 1-amino-2-(3, 4-dihydroxyphenyl)-ethylphosphonic acid and distribution studies in mice bearing the Harding-Passey melanoma

The aminophosphonic acid analogue of DOPA, DL-1-amino-2-(3,4-dihydroxy-phenyl) ethylphosphonic acid (ADEP) has been synthesised. The compound was of low toxicity; a single dose of 2 g/kg given to mice subcutaneously was not lethal.[³H]ADEP was injected subcutaneously into mice carrying the established Harding-Passey melanoma, and the distribution of the tritium determined. The highest initial concentration of radioactivity was in the kidneys, adrenal glands and eyes. Isotope content fell to low values in all tissues within 8 days or less but the tumour retained radioactivity for a longer period than did the other tissues examined.ADEP served as a substrate for mushroom tyrosinase.     

Synthesis, processing, and secretion of the Marek''s disease herpesvirus A antigen glycoprotein.

The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption.     

RecA independent recombination of poly[d(GT)-d(CA)] in pBR322.

Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly[d(GT)-d(CA)] carried in a derivative of pBR322 were recombinogenic in a recA host. Recombination brought about by poly[d(GT)-d(CA)] tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly[d(GT)-d(CA)] tracts, recombination also occurred between sequences bordering the dinucleotide repeats. (ii) recombination was enhanced when two poly[d(GT)-d(CA)] tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs. The mechanism by which poly[d(GT)-d(CA)] stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E. coli may involve formation of Z-DNA.     

Pulmonary toxicity of 1,1-dichloroethylene: correlation of early changes with covalent binding

Administration of a single intraperitoneal dose of 1,1-dichloroethylene (125 mg/kg, 1,1-DCE) to mice resulted in bronchiolar injury with selective necrosis of Clara cells. Degenerative changes were manifest in Clara cells as early as 1 h following 1,1-DCE exposure, and were characterized by marked swelling of mitochondria and aggregation of chromatin against the nuclear membrane. Cell death was apparent at 2 h; by 8 h, areas of the bronchiolar epithelium were devoid of lining cells, and at 24 h, the majority of Clara cells were exfoliated. The residual epithelium consisted of flattened cells which formed a thin lining for the airway. Necrosis of Clara cells early in the course of 1,1-DCE exposure coincided with peak covalent binding of [14C]1,1-DCE and significant depression of components of the pulmonary mixed-function oxidase system; cytochrome P-450 and aryl hydrocarbon hydroxylase activity were markedly reduced but not depleted. Liver damage involving centrilobular hepatocytes was observed at 24 h in 30% of treated animals, and coincided with significant inhibition of aryl hydrocarbon hydroxylase activity; cytochrome P-450 content, however, remained unchanged. While changes in the liver evoked by 1,1-DCE were less striking, the results in lung demonstrate positive temporal correlations between structural damage, peak covalent binding and disturbances of monooxygenase enzymes.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Conformational flexibility of the regulatory site of phosphoribulokinase as demonstrated with bifunctional reagents.

Phosphoribulokinase (PRK) is one of several chloroplastic enzymes whose activity is regulated by thiol-disulfide exchange via thioredoxin. Activation entails reduction of an active-site disulfide bond between Cys16 and Cys55. Bifunctional cross-linking reagents have been used to approximate the interresidue distance between Cys16 and Cys55, an issue which impinges on the relative conformational states of the activated and deactivated forms of the enzyme. Spinach PRK is rapidly inactivated by stoichiometric levels of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (FNPS) or 1,5-difluoro-2,4-dinitrobenzene (DFNB), which span 9 and 3.5 A, respectively. ATP, but not ribulose 5-phosphate, retards the rate of inactivation, suggesting that modification has occurred at the nucleotide binding domain of the active site. Sulfhydryl modification is indicated by partial reversibility of inactivation as effected by exogenous thiols. Tryptic mapping by reverse-phase chromatography of [14C]carboxymethylated enzyme, subsequent to its reaction with either FNPS or DFNB, demonstrates modification of Cys16 and Cys55 by both reagents, and formation of only one major chromophoric peptide in each case. On the basis of the sequence analysis of the purified chromophoric peptides, Cys16 and Cys55 are cross-linked by both FNPS and DFNB. Thus, the intrasubunit distance between the beta-sulfhydryls of Cys16 and Cys55 is dynamic rather than static. Diminished conformational flexibility upon oxidation of the regulatory sulfhydryls to a disulfide may be partially responsible for the concomitant loss of enzymatic activity.