Prevention of the normal expansion of maternal plasma volume: a model for chronic fetal hypoxaemia

The effects of inadequate expansion of maternal blood volume on uterine blood flow, fetal oxygen levels and vasoactive mediators during the third trimester were studied in 8 pregnant sheep. Results were compared to those obtained during 15 normal pregnancies. Prevention of the normal (20 ml/day) increase in maternal plasma volume was achieved by repeated haemorrhage and injections of furosemide. These treatments also reduced the rise in blood flow to the pregnant uterine horn that normally occurs during this period of gestation: at term flow was only 508 +/- 61 (SEM) compared to 838 +/- 83 ml/min in the control group (P greater than 0.01). This reduction in uterine blood flow caused a gradual fall in fetal PaO2, and rise in fetal levels of plasma renin activity, vasopressin, catecholamines and angiotensin II without change in pHa or base excess. Four to 5 days prior to delivery, the difference from control in PaO2 was -3.9 +/- 0.5 mmHg, plasma renin activity +2.9 +/- 1.7 ng/ml.h, vasopressin +4.2 +/- 1.1 pg/ml, catecholamines +957 +/- 145.3 pg/ml and angiotensin II +243 +/- 108.2 pg/ml. Furthermore, the fall in PaO2 and rise in vasoactive mediators that normally occur 3-5 days prior to the onset of labour was either absent (PaO2 and plasma renin activity) or blunted. Thus when expansion of blood volume during pregnancy is inadequate, blood flow to the uterus is adversely affected. This leads to various degrees of chronic fetal hypoxaemia and stimulation of vasoactive mediator systems. However, the normal stimulation of vasoactive mediator systems that occurs 3-5 days before delivery appears to be blunted. Experimental prevention of blood volume expansion during pregnancy produces an excellent model for the study of chronic mild fetal hypoxaemia.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Photoreceptor-specific efficiencies of β-carotene,zeaxanthin and lutein for photopigment formation deduced from receptor mutant Drosophila melanogaster

Summary Drosophila rearing media had only -carotene, zeaxanthin or lutein as precursors for photopigment chromophores. Zeaxanthin and lutein are potentially optimum sources of the 3-hydroxylated retinoids of visual and accessory photopigments. Mutants made the electroretinogram in white (w) eyes selective for compound eye photoreceptors R1–6, R7 and R8: R1–6 domiantes w's electroretinogram; R7/8 generates w;ora's (ora = outer rhabdomeres absent); R8 generates w sev;- ora's (sev = sevenless). Microspectrophotometry revealed R1-6's visual pigment. In w, all 3 carotenoids yielded monotonic dose-responses for sensitivity (Fig. 4) or visual pigment (Fig. 7). An ultraviolet sensitivity peak from R1-6's sensitizing pigment was present at high but not low doses (Fig. 1). In w;ora, all 3 carotenoids gave similar spectra dominated by R7's high ultraviolet sensitivity (Fig. 2). For w sev;ora, all spectra were the shape expected for R8, peaking around 510 nm (Fig. 3). The sensitivity dose-response was at its ceiling except for low doses in w;ora (Fig. 5) and zero supplementation in w sev;ora (Fig. 6). Hence, without R1-6, most of our dose range mediated maximal visual pigment formation. In Drosophila, -carotene, zeaxanthin and lutein mediate the formation of all major photopigments in R1-6, R7 and R8.Abbreviations ERG electroretinogram - MSP microspectrophotometry - HPLC high pressure liquid chromatography - n.a. numerical aperture - w, sev, ora Drosophila mutants - y, p, r marg types of R7 and R8     

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.     

A laser-T-jump study of the adsorption of dipolar molecules to planar lipid membranes

The adsorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as well as of other dipolar molecules to the interface of artificial lipid membranes gives rise to a change of the dipole potential between the membrane interior and water. As a consequence of the adsorption of the neutral species, the conductance of planar membranes, observed in the presence of the macrocyclic ion carriers nonactin or valinomycin, may change by many orders of magnitude. Using this effect in combination with a laser-T-jump technique, the kinetics of the adsorption process were measured and were interpreted on the basis of a Langmuir-isotherm. A partition coefficient (at small concentrations) of _HA =4.7·10^–4 cm, a rate constant of desorption k _HA100 s^-1 and a maximum surface density N _D=7.7·10¹³/cm² were found. The concentration at half saturation is K _HA=2.7·10^-4 M.Using these values the membrane conductance induced by the ion carrier nonactin and the shape of the current-voltage relationship as a function of the ligand concentration in water was analyzed. A maxiumum dipole potential of V _D ^max =-239 m V and a contribution of b=3.1·10^-15V cm² per single adsorbed 2,4-D molecule was found. 74% of the dipole potential acts on the inner membrane barrier separating the two interfacial adsorption planes of nonactin. The remainder (26%) favours interfacial complex formation between nonactin and K⁺ from the aqueous phase. The data hold for membranes formed from dioleoyllecithin in n-decane.     

Control of inspiratory duration in premature infants

We used single-breath mechanical loads and airway occlusions in premature infants to determine whether maturation influences the reflex control of inspiratory duration. We measured flow, volume, airway pressure, and surface diaphragmatic electromyogram (EMG) in 10 healthy preterm infants [33 +/- 1 (SD) wk gestation], 2-7 days of age. Three resistive and two elastic loads and occlusions were applied to the inspiratory outlet of a two-way respiratory valve. Application of all loads resulted in inspired volumes significantly decreased from control (P less than 0.001), and these decreases were progressive with increasing loads. Inspiratory duration (TI) was prolonged from control by all loads and occlusions when measured from the diaphragmatic EMG (neural TI) and by all but the smaller elastic load when measured from the flow tracing (mechanical TI). Similar decreases in inspired volume at the end of neural TI produced by application of both elastic and resistive loads resulted in comparable prolongation of neural TI. In contrast, for comparable volume decrements, resistive loading prolonged mechanical TI more than elastic loading (P less than 0.001). Mechanical and neural TI values of the breath after the loaded breath were unchanged from control values. Comparison of the neural volume-timing relationship in premature infants with our data in full-term infants suggests that the strength of the timing response to similar relative decrements in inspired volume is comparable. We conclude that reflex control of neural TI in premature infants depends on the magnitude of inspired volume and is independent of the volume trajectory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA.

Rabbit serum samples from eleven different research facilities were evaluated for the presence of immunoglobulin G against Pasteurella multocida by using an enzyme-linked immunosorbent assay (ELISA). Each facility which submitted serum samples also provided a brief history of each rabbit colony tested. Rabbits from colonies reported to have endemic P. multocida or of undetermined status had 83 (58.9%) of 141 rabbits that were positive. Colonies reported to be free from P. multocida had 110 (92.4%) of 119 rabbits that were negative by ELISA. The ELISA test described here showed a high degree of agreement (92-94%) with two other P. multocida ELISAs at different diagnostic facilities. This study confirms that an ELISA testing for serum antibodies against the P. multocida is a reliable diagnostic tool to screen colonies for P. multocida.