Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size,and for reductions in membrane surface area and immature granule volume during granule maturation

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 unit progranules of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures.     

Vascular Development and Sap Flow in Apple Pedicels

Xylem and phloem tissues of the pedicel of apple fruit increasein cross-sectional area throughout development. The increasein phloem is similar in the two cultivars examined (Cox's OrangePippin and Royal Gala) and reflects a steadily increasing phloemsap flow to the fruit. The increase in xylem tissue is due toa proliferation of non-conducting, structural, components sinceclose examination reveals no increase in the number of vesselelements from just after flowering onwards. The greater number,and the larger diameter, of the vessels in Cox's explains theinitially higher xylem conductance found in this cultivar. In vitro measurements of xylem exudation reveal a decline duringthe growing season in the xylem conductance of both cultivarsand an increasing proportion of fruit (particularly in Cox's)in which the xylem comes to be totally non-conducting. Thisobservation is in line with previously reported measurementsof xylem sap flow in vivo. The straightforward techniques used in this study offer a feasiblealternative to more arduous methods of assessing xylem and phloemsap flows and their balance during growth.Copyright 1994, 1999Academic Press Apple, xylem, phloem, vascular development, sap flow, Malus domestica Borkh     

Machado-Joseph Disease in Pedigrees of Azorean descent is Linked to Chromosome 14

A locus for Machado-Joseph disease (MJD) has recently been mapped to a 30-cM region of chromosome 14q in five pedigrees of Japanese descent. MJD is a clinically pleomorphic neurodegenerative disease that was originally described in subjects of Azorean descent. In light of the nonallelic heterogeneity in other inherited spinocere-bellar ataxias, we were interested to determine if the MJD phenotype in Japanese and Azorean pedigrees arose from mutations at the same locus. We provide evidence that MJD in five pedigrees of Azorean descent is also linked to chromosome 14q in an 18-cM region between the markers D14S67 and AACT (multipoint lod score +7.00 near D14S81). We also report molecular evidence for homozy-gosity at the MJD locus in an MJD-affected subject with severe, early-onset symptoms. These observations confirm the initial report of linkage of MJD to chromosome 14; suggest that MJD in Japanese and Azorean subjects may represent allelic or identical mutations at the same locus; and provide one possible explanation (MJD gene dosage) for the observed phenotypic heterogeneity in this disease.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Terminal electron transport system (ETS)-activity in the sediment of Lake Balaton,Hungary

Terminal electron transport system (ETS)-activity of the sediment and plankton of Lake Balaton, the largest shallow lake of Central Europe was measured by tetrazolium-reduction biweekly during 1989–1990 and in the spring of 1991. Sediment proved to be enzymatically active to 30-35 cm down in the hypertrophic Keszthely Bay and to 15–20 cm down in the meso-eutrophic Siófok Basin. Sediment ETS-activity exceeded planktonic activity 15 to 24 fold.The total activity m^–2 showed one or two order of magnitude higher respiratory potential in Lake Balaton than needed for complete oxidation of the planktonic primary production; most of this potential was detected in the upper 3–5 cm sediment layer in springs. Incubations of cell-free homogenates of sediment bacteria showed that ETS remains active days after death of organisms at low temperature. Accumulated postmortem ETS-activity derived from the benthic diatoms, bacteria, plankton deposit and dead summer macrophytes seems to be responsible for the high ETS-activity of the sediment in the warming periods in springs. These enzyme fractions may contribute to the rapid oxidation of the alkaline, well-aerated lake.     

Ern?hrungsphysiologie des Blaunackenmausvogels (Urocolius macrourus pulcher)

Zusammenfassung Beim Blaunackenmausvogel (Urocolius macrourus pulcher) wurde die Ausnutzung der verschiedenen Nährstoffe (Fette, Eiweiße, Kohlenhydrate) und die Assimilationseffizienz des Gesamtfutters untersucht. Mausvögel sind Vegetarier und hauptsächlich frugivor. Im Versuch erhielten die Vögel vier Diäten: I. Mischfutter (Bananen, Salat, gekochtes Ei, gekochter Reis, Apfel); II. Bananen; III. Fruchtfutter (Pfirsiche und Birnen); IV. Eiweißfutter (Quark und gekochtes Weißei). Die minimalen Darmpassagezeiten betrugen für alle vier Diäten zusammengefaßt zwischen 6 und 18 min. Damit kommen die Mausvögel an die Werte für Nektarfresser heran (unter 15 min). Der Darmtrakt ist mit 19,0 ±2,4 cm (n = 16) für einen Vogel dieser Körpermasse ( 56,4 ±2,7 g; 49,2 ±2,9 g) extrem kurz und zeigt den für Fruchtfresser typischen einfachen Bau. Blinddärme fehlen. Die mittlere Gesamteffizienz für die vier Diäten liegt bei 71,0 %. Die Werte variieren in einem relativ engen Bereich zwischen 65,9 % (Banane) und 74,0 % (Mischfutter). Damit sind Blaunackenmausvögel innerhalb der Vögel als relativ schlechte Nahrungsverwerter zu bezeichnen. Für Fruchtfresser allgemein werden allerdings noch niedrigere Werte zwischen 30 % und 70 % angegeben. Die Assimilationsrate der einzelnen Nährstoffe hängt in starkem Maße vom Angebot im Futter ab. Die Zusammensetzung der assimilierten Nahrung zeigt, daß Kohlenhydrate mit 89,0–91,1 % den weitaus größten Anteil am Energiestoffwechsel haben. Vermutlich werden nur gelöste oder leicht verdauliche Einfachzucker bzw. Stärken verwertet. Zellulose wird praktisch ungenutzt ausgeschieden. Fette und Proteine spielen unter Normalbedingungen nur eine untergeordnete Rolle. Der Energieumsatz ist nahrungsabhängig und beträgt 62,5 J/g·h bei der Bananendiät, 69,2 J/g·h bei der Fruchtdiät, 87,3 J/g·h bei der Eiweißdiät und 99,6 J/g·h beim Mischfutter.

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Nutrient physiology of the Blue-naped Mousebird (Urocolius macrourus pulcher)

Blue-naped Mousebirds were fed with four different diets: I. mixed food (bananas, salad, boiled eggs, boiled rice, apples); II. bananas only; III. soft fruits (pears and peaches); and IV. food enriched with protein (curd cheese and egg-white). The minimal times for food passage through the digestive tract were 6–18 min altogether. This is similar to the data known from nectarivorous birds (<15 min). The intestines are extremely short (19,0 ±2,4 cm; n = 16) and simple-structured, without specialization and without any caeca. The overall efficiency for the diets with 71,0 %, ranging between 65,9 % (bananas) and 74,0 % (mixed food), is relatively low. However, for frugivorous birds in general, far lower efficiencies are recorded (30–70 %). The assimilation efficiencies of nutrients depend on their amount in food and the physiological and seasonal requirements. The composition of the assimilated food shows that carbohydrates, having the largest part with 89,0–91,1 %, are most important for energy supply. Presumably, the birds utilize only sugars being easy to digest, because cellulose is removed. Fat and protein are playing a subordinate role in metabolism. The metabolic turnover differs with the diet and ranges between 62,5 J/g·h (bananas), 69,2 J/g·h (fruits), 87,3 J/g·h (protein food) and 99,6 J/g·h (mixed food).

     

Degradation by and toxicity to bacteria of chlorinated phenols and benzenes,and hexachlorocyclohexane isomers

Mixed cultures degrading chlorinated benzenes, chlorinated phenols, or hexachlorocyclohexane (HCH) as the sole source of carbon and energy were obtained by enrichment from contaminated soil samples. Cultures which metabolized 3-chlorophenol (3-CP), 2,3-dichlorophenol (2,3-DCP), or 2,6-dichlorophenol (2,6-DCP) were able to utilize several other chlorinated compounds as substrates, whereas cultures enriched with 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB), -HCH, or -HCH did not metabolize most of the other chlorinated congeners tested. Chloride release and growth rates with all four chlorinated phenols decreased with increasing initial substrate concentrations within the range of 30–250 mol liter^–1. Maximum chloride release was 3.8 mg liter^–1 corresponding to 35 mol liter^–1 trichlorophenol within 7 weeks. In contrast, the rate of metabolism of the nonphenolic compounds 1,2,4,5-TeCB, -HCH, or -HCH increased with increasing substrate concentrations. Initial concentrations of 750 mol liter^–1 -HCH or 1,2,4,5-TeCB were completely dechlorinated within 2 weeks. Because aqueous solubility and bioavailability of the chlorophenolic compounds is much higher than that of the nonphenolic compounds, it is suggested that the high bioavailability of the chlorophenolic compounds is the reason for the high toxicity of these substrates to the degrading cultures. In contrast, the low aqueous solubilities of the chlorinated benzenes and HCH-isomers caused consistently low concentrations in the medium, which were high enough to induce degradation but too low to damage the bacterial cells.Correspondence to: E. Lang     

Interfacial and micellar behavior of glucose lipid

Summary Glucose lipid of notable surfactant properties was produced by using hydrocarbon assimilating bacterium of the bacterial strain MM1. Its surface active properties were notable in spite of ,-hydrophilic and bulky molecule. The critical micelle concentration (cmc) was small (0.165 M); surface and interfacial tension for hexadecane at 0.1 % (pH 7.35) solution, 24.6 and 13.3 mN/m, respectively. The emulsifying action was excellent and comparable to those of rhamnolipids. The micelle has the outer radius. 28.5 Å and inner core, 6.7 Å on the basis of the concentric spherical shell model by means of the small angle X-ray solution scattering measurement.Dedicated to Professor Fritz Wagner on the occasion of his 65th birthday.