Microbial reductions of the sesquiterpene quadrone

One hundred microorganisms have been screened for their abilities to selectively modify the structure of the sesquiterpene lactone known as quadrone. The only products obtained were those formed when the 4-ketone functional group was reduced to the stereoisometric 4-quadronols. Quadrone alcohol isomers of (S) or (R) absolute configurations were identified by proton and carbon n.m.r., and high performance liquid chromatography (h.p.l.c.) was used to separate and quantitate these compounds in extracts of fermentations. Microorganisms were categorized according to their abilities to achieve Re- or Si-face carbonyl reduction to yield (S)- or (R)-alcohol isomers by h.p.l.c. Three groups of microorganisms were identified: those yielding only the (R)-alcohol isomer; those yielding only the (S)-alcohol isomer; and those providing mixtures of the two alcohol isomers. With quadrone as substrate, Mucor and Curvularia spp. may contain either Re- or Si-face reductases. The selection of microorganisms for their abilities to achieve enantiospecific reductions of ketones to alcohol products is discussed.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

Summary The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combintion, of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10⁻⁵ M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. This work was supported in part by Grants CA-07175, CA-22484, and CA-17334 from the National Cancer Institute. D. P. G. is a Predoctoral Fellow at the Food Research Institute, supported by a fellowship from the Monsanto Fund and by NIH Grant R01-AI 15693 to Prof. Michael W. Pariza, Food Research Institute, University of Wisconsin, Madison.     

Entry of immotile spermatozoa into zona-free hamster ova

We studied six men whose spermatozoa were immotile and possessed a variety of sperm tail structural abnormalities by electron microscopy. The semen of all six subjects had a normal percentage of oval forms and sperm undergoing capacitation and acrosome reaction. Despite the absence of motility, when incubated sperm from these subjects was added to a microdrop of medium containing zona pellucida-free hamster ova, sperm penetration or entry into the cytoplasm of from 1–9% of the eggs was evident with phase contrast microscopy. This latter finding suggests that, at least in this system, oocytes actively facilitate sperm incorporation. Penetration was absent when sperm of fertile men were rendered immotile, though still viable, by heat treatment.     

Influence of the concentration of sperm on the percentage of eggs fertilized for three strains of mice

This study was conducted to determine the optimal concentration of sperm to use for the insemination of females to detect differences among strains of mice in the percentage of eggs fertilized. Female ICR mice were inseminated with sperm of concentrations ranging from 0.25 to 8 × 10⁶/50 μl from males of either DBA/2N, CF1, or C57BL/6N strains. Differences among strains were detected only when approximately 50% of the eggs were fertilized but not when each of the strains fertilized either a high or low percentage of eggs. The optimal concentration of sperm therefore was the concentration that gave approximately 50% fertilized eggs.     

Normal levels of zinc and sulfhydryls in morphologically abnormal populations of spermatozoa from moderately zinc-deficient rats

Zinc is required for spermatogenesis in mammals and is concentrated in the dense outer fibers of the sperm tail, where it is associated with cysteine-rich protein. To investigate the effects of marginal zinc deficiency upon dense fiber formation and upon sperm quality in general, weanling Sprague-Dawley rats were administered a commercial low-zinc diet, supplemented with phytate, for approximately 60 days, and were compared with controls fed the same diet plus 50 ppm zinc in their drinking water. The following characteristics of the zinc-deficient rats were significantly lower than in the controls: body weight, testis weight, epididymis weight, seminal vesicle weight, sperm content of the cauda epididy-midis, sperm motility, testis zinc, and hair zinc. By contrast, the levels of sperm zinc and sperm sulfhydryls were the same in the zinc-deficient and control rats. The zinc-deficient rats displayed a highly variable spectrum of sperm defects, which included decapitation, disorganized and redundant tail elements, and superfluous cytoplasm. However, abortive dense fiber development was only rarely observed. Apparently, even when availability of zinc is limited and reduced sperm production ensues, elaboration of dense fibers rich in zinc and sulfhydryls continues to be obligatory for the completion of spermiogenesis.     

Interrelationships between water and cellular metabolism inArtemia cysts

Cysts of the crustaceanArtemia are a useful model for studies on intracellular water because they are capable of essentially complete and reversible desiccation. We have used a variety of techniques on this system, the present work being an attempt to estimate the density of intracellular water (ρ_w). The density of individual cysts was evaluated from sedimentation velocity. Heptane displacement methods were used to determine the volume of a known mass of cysts, from which the density was calculated. The two methods produce comparable results. It was shown that the densities and water contents of large masses of cysts accurately reflect those of individual cysts. Cyst densities (ρ_c) were determined over the entire range of water content from 0 to 0.63 weight fraction of water (W _f), and temperature dependence was measured for 0.61W _f over 2–41°C. The following refer to 25°C. No marked change was detected in ρ_c until the water content exceeded 0.15W _f, at which ρ_c decreased as a linear function of Wf to maximum water content. However, the cyst does not behave ideally in the sense that the densities of the nonaqueous components and added water are not additive as a function ofW _f. The partial specific volume of water in cysts at maximum hydration was estimated to be 3% larger than that of pure water. These observations are compared with density measurements on other systems, and with previous findings on the physical properties of water in this system.     

Sex-related differences in first-stage impedance cardiographic variables

Impedance cardiography (ZCG), a noninvasive technique used to determine stroke volume and ventricular performance, is currently being utilized more extensively in psychophysiological research and may also prove a useful tool in the control of hypertension. To date, most studies employing ZCG have tested male subjects or patients. However, women and men differ on two important parameters used to calculate stroke volume: thoracic impedance (Zo), and the first derivative of change in impedance over time (dZ/dt_min). Examination of the clinical records of 19 females and 19 males, all nonmedicated, indicated that women had higher Zo levels (F(1, 36)=46.2,p<.0001) and higher dZ/dt_min levels (F(1, 36)=51.8, p<.0001), although calculated stroke volume indexes did not differ. A second sample of 10 women and 8 men, all healthy and nonmedicated, confirmed these findings. The differences in Zo are not due entirely to sex differences in height, weight, percent body fat, and subscapula skin-fold thickness, although these factors differ across sexes. It is important for researchers and clinicians utilizing ZCG with female subjects to be aware of these differences.     

Sequence comparison of rat liver phenylalanine hydroxylase and its cDNA clones

Classical phenylketonuria, an inborn error in metabolism, is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. The identification of putative cDNA clones coding for rat liver phenylalanine hydroxylase by hybrid-selected translation has previously been reported [Robson, K. J., Chandra, T., MacGillivray, R. T. A., & Woo, S. L. C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4701-4705]. The authenticity of the clones, however, could not be definitively ascertained at the time because of a lack of amino acid sequence data of the enzyme in the literature. Purified rat liver phenylalanine hydroxylase was subjected to cyanogen bromide treatment, and the resulting fragments were used for N-terminal amino acid sequence analysis. The partial amino acid sequence was then compared to that deduced from an open reading frame in the nucleotide sequence of the cDNA clones. A perfect match of 17 amino acid residues was found between the two sequences following a unique methionine codon present in the nucleotide sequence, thereby providing unambiguous evidence for the identity of the rat liver phenylalanine hydroxylase cDNA clones.