Production of cloned carboxypeptidase G2 by Escherichia coli: Genetic and environmental considerations

Summary The optimum production of cloned carboxypeptidase G₂ from plasmid pNM21 byEscherichia coli was found to be strongly strain- and temperature-dependent. The superior host was strain RV308 and the preferred growth temperature 28°C. Copy number, which decreased during exponential growth of all strains examined, did not relate in these studies to the level of enzyme production: the strain with the highest enzyme yield also having the lowest overall copy number.     

Effects of dihydroergotoxine (hydergine) on peripheral blood alcohol levels

To examine the effects of dihydroergotoxine (DHET) on peripheral blood ethanol levels of adult female mice, ethanol (2 g/kg) was administered orally, either alone or in combination with DHET (2, 4, or 8 mg/kg). Blood was drawn after the first day of drug administration (acute study) and again after 21 days (chronic study). Two additional groups of mice received a single dose of ethanol (2 g/kg) and DHET (2 mg/kg) 15 minutes apart, one group receiving ethanol first, the other DHET as the first dose. Blood samples (100μ1) were collected from the tail vein at 5, 15, 30, 60, 120 and 180 minutes after treatment. Samples were analyzed by headspace gas chromatography. Results indicate that (a) combining ethanol with DHET significantly reduced blood ethanol levels compared to administration of ethanol alone, (b) chronic conditions produced higher blood ethanol levels, and (c) administration of ethanol 15 minutes before DHET produced a 27% lowering of peripheral blood alcohol levels compared to the reverse order of administration. These data suggest that DHET may be useful for alleviating some of the symptoms associated with alcohol intoxication.     

LATERAL MANDIBULAR WALL THICKNESS IN TURSIOPS TRUNCATUS: VARIATION DUE TO SEX AND AGE

In odontocetes the mandibular bone serves two functions: to capture prey, and as a means of the reception and transmission of sound waves through a fat body in the mandibular canal, which opens posteriorly as the mandibular foramen. The posterior part of the lateral wall of the odontocete mandible is thin, and appears to represent a compromise between a strong mandible for prey capture and a thin vibrating plate for hearing. We studied the intraspecific variation of minimum thickness of the lateral mandibular wall along four transects (T1-T4) at the area of the mandibular foramen, in relation to the skull size and the mandibular size in different-aged bottlenose dolphins, Tursiops truncatus (18 females between 1 and 42 yr, 17 males between 1 and 32 yr). The minimum thickness was absolutely at its lowest at the most posterior transect T1, but did not vary significantly between the sexes or between the ages. The minimum thickness varied significantly at the two most anterior transects, T3 and T4, both between the sexes and among the ages. The thickness increased throughout life among males, whereas in females it first increased and then starts to decrease around the age of 20.     

Responses of a eutrophic pond community to separate and combined effects of N:P supply and planktivorous fish: a mesocosm experiment

We conducted an outdoor mesocosm experiment of factorial design consisting of three levels of nutrient supply (no nutrient addition and additions of nitrogen and phosphorus in ratios of 10:1 and 45:1) cross-classified with two levels of bluegill (Lepomis macrochirus) (presence and absence). Nutrient supply significantly affected total phosphorus (TP), total nitrogen (TN), TN: TP ratio, turbidity, Secchi depth, phytoplankton chlorophyll, filamentous blue-green algae, periphyton chlorophyll, Asplanchna and non-predatory rotifers. The presence of bluegill significantly increased TP, turbidity, diatoms, unicellular green algae, colonial blue-green algae, filamentous blue-green algae, periphyton chlorophyll, Asplanchna and non-predatory rotifers, and decreased Secchi depth, cladocerans, cyclopoid copepodids, copepod nauplii and chironomid tube densities. Nutrient supply and fish effects were not independent of each other as shown by significant nutrient × fish interaction effects for TP, Secchi depth, filamentous blue-green algae, periphyton chlorophyll, Asplanchna and non-predatory rotifers.     

The characterization of energized and partially de-energized (respiration-independent) β-galactoside transport into escherichia coli

Unidirectional fluxes of [¹⁴C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN^?) conditions are analyzed kinetically.When the cells are energized, the value for $\text{V}$ influx is $\text{0.45 ± 0.01}\text{mM}$ internal concentration increment/s and $\text{K}{}_{\mathrm{t}}$ is $\text{0.26 ± 0.03}\text{mM}$. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is $\text{2 · 10}{}^3$.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of $\text{8.8 ± 0.2 · 10}{}^{\mathrm{?4}}\text{s}{}^{\mathrm{?1}}$.The transport in the presence of CN^? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O₂).The effects of CN^? are to lower the $\text{V}$ for influx and to change the efflux from a non-saturable to a saturable process with a value for $\text{K}{}_{\mathrm{t}}$ (60 mM) intermediate between that for energized efflux ($\text{> 250}\text{mM}$) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.     

A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

We suggest a method of quantitating the motile actions of surface protrusions in spreading animal cells in culture. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. Studying 3T3 cells with this assay, we found that the presence of Na+, K+, Cl-, and Mg++ or Ca++ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the cells for at least 50 min. Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of three glucose analogues which are generally believed to be nonmetabolizable cannot restore the activity. Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Furthermore, serum codetermines which of the major surface extension, filopodia, lamellipodia, or lobopodia, is predominantly active. We found three distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B or the particle removal are very similar in all three cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal threefold.     

Distribution of apolipoproteins A-I and B among intestinal lipoproteins

Chylomicrons and very low density lipoproteins (VLDL) are produced by the intestine and these nascent particles are thought to be similar to their counterparts in intestinal lymph. To study the relationship between these lipoproteins within the cell and those secreted into the lamina propria and lymph, we have isolated enterocytes, lamina propria, and mesenteric lymph from rats while fasted and after corn oil feeding. Apolipoprotein A-I and B content were measured by radioimmunoassay in cell, lamina propria, and lymph fractions separated by Sepharose 6B and 10% agarose chromatography, and by KBr isopycnic density centrifugation. ApoA-I in the cell and the underlying lamina propria was found partly in those fractions in which chylomicron and very low density lipoproteins (chylo-VLDL) and high density lipoproteins (HDL) elute, but more abundantly where unassociated 125I-labeled apoA-I was eluted. In the lymph, however, 74% of apoA-I eluted in the HDL region and no peak of free apoA-I was found. ApoB and apoC-III within the enterocyte were found distributed in the position of particles eluting not only with chylomicrons and VLDL, but also in the regions corresponding to LDL and HDL. In the lamina propria and lymph, on the other hand, most of the apoB was found in the region of VLDL and chylomicrons. These results indicate that the patterns in lymph lipoproteins and the lamina propria do not exactly mirror the distribution of apoA-I and B among lipoproteins inside the cell. This may be because intracellular apoproteins may be unassociated with lipoproteins, or they could be associated with lipoproteins in various stages of assembly of protein with lipids. Furthermore, the apoprotein composition of intestinal lipoproteins is altered after secretion from the enterocyte. Finally, not all apoproteins seem to be secreted in association with identifiable lipoprotein particles from the enterocyte.     

Molecular cloning and nucleotide sequence of deer papillomavirus.

The genome of deer papillomavirus (DPV) isolated from American white-tailed deer was cloned into pBR322, and the entire nucleotide sequence of 8,374 base pairs was determined. The overall genetic organization of the DPV genome was similar to that of other papillomaviruses. All significant open reading frames were located on one strand, and the locations of putative promoters and polyadenylation signals were similar to those identified in the closely related bovine papillomavirus type 1 (BPV-1) genome. The DPV genome was approximately colinear with BPV-1 except for a noncoding region separating the early and late regions. The regions of highest nucleotide sequence homology between DPV and BPV-1 were found in the E1 open reading frame coding for BPV-1 DNA replication function and in the L1 open reading frame, which encodes the major capsid protein of BPV-1.     

Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia.

A small-for-gestational-age female infant born at term developed severe lactic acidosis and died on day 13 of life. Two previous sibs had also died of overwhelming lactic acidosis in the neonatal period. The lactate-to-pyruvate and 3-hydroxybutyrate-to-acetoacetate ratios were elevated at 136 and 42 to one, respectively. The activities of the pyruvate dehydrogenase complex and pyruvate carboxylase in cultured skin fibroblasts were normal but a defect in respiration was indicated by the low rates of conversion of 1-[14C]pyruvate, glutamate, and lactate to 14CO2 in these cells. Skin fibroblast cultures also displayed an elevated lactate-to-pyruvate ratio (72:1) when incubated with glucose as substrate compared to control cell cultures (20:1). When mitochondrial preparations of skin fibroblasts (prepared by digitonin extraction) were tested for their ability to synthesize ATP from a variety of substrates, it was found that those of the patient made adequate amounts of ATP with either succinate or ascorbate/tetramethyl-phenylenediamine as substrate but not with the NAD-linked substrates pyruvate, isocitrate, and palmitoyl carnitine. We propose that this is indicative of a defect in the respiratory chain between NADH and coenzyme Q, for the first time demonstrable in cultured skin fibroblasts.