Infectivity of Cryptosporidium parvum oocysts following cryopreservation.

This study was undertaken to investigate the cryopreservation of Cryptosporidium parvum oocysts. Oocysts purified from mouse feces were suspended in distilled water, 10% glycerin, and 2.5% potassium dichromate. They were stored at -20 C and -80 C for 2, 7, and 30 days, respectively. In addition to the purified oocysts, the feces of C. parvum-infected mice were preserved under the same conditions described above. Purified and fecal oocysts were thawed at 4 C, and their viability was assessed by a nucleic acid stain, excystation test, tissue culture infectivity test, and infectivity to immunosuppressed adult mice. Oocysts purified from fecal material prior to cryopreservation lost most of their viability and all of their infectivity for tissue culture and mice. However, when oocysts were cryopreserved in feces, between 11.7 and 34.0% were judged to be viable and retained their infectivity for mice when stored at -20 C (but not -80 C) for 2, 7, and 30 days. Clearly, fecal material provides a cryoprotective environment for C. parvum oocysts stored at -20 C for at least 30 days.     

Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.     

Antigen expression from the ribosomal DNA repeat unit of Giardia intestinalis.

The amitochondrial human intestinal parasite Giardia intestinalis is regarded to be the most ancient living example of single-celled eukaryotes and should display primitive features of pre-metazoan gene regulation. Characterization of E. coli clones which express Giardia antigens from plasmid vectors has revealed that an antigen is encoded by the rDNA repeat unit from the strand complementary to that encoding the rRNAs. The open reading frame (ORF) originates in the spacer region between the small (SS) and large (LS) subunit rRNA genes and terminates within the LS rRNA gene. The promoter region of this ORF has characteristics of both RNA polymerase (pol) II and pol III regulatory sequences, suggestive of gene regulation before these different promoter types evolved. The rDNA repeat unit is located on multiple chromosomal sites which are different in each isolate, although the electrophoretic karyotypes appear very stable in Giardia from both human and animal sources.     

The Kok Effect in Chlamydomonas reinhardi

A Haxo-Blinks rate-measuring oxygen electrode together with a modulated light source gave an average current signal (change in net O₂ exchange) and a modulated current signal (photosynthetic O₂ evolution). Using this apparatus, net O₂ exchange and photosynthetic O₂ evolution at low intensities have been studied in the green alga, Chlamydomonas reinhardi. At both 645 nm and 695 nm, the curves of net O₂ exchange as a function of light intensity were steeper at lowest intensities than about compensation, indicative of the Kok effect. The effect was greater at 695 nm than at 645 nm. The corresponding curves of photosynthetic O₂ evolution, on the other hand, showed no Kok effect; here, the slope was lowest at lowest intensity. The absence of the Kok effect in O₂ evolution, together with its sensitivity to monofluoroacetic acid, show that it is due to an interaction of photosynthesis and respiration. The effect was exaggerated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In the presence of concentrations of this inhibitor sufficient to inhibit O₂ evolution completely, a light-induced change in net O₂ exchange remained. This was interpreted as a system I dependent depression of respiratory O₂ uptake. The Kok effect remained undiminished in concentrations of carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol which partially uncoupled either oxidative phosphorylation alone or both oxidative and photosynthetic phosphorylations. The above results can be explained within a model of the Kok effect in which O₂ uptake is depressed by diversion of reductant away from respiratory electron transport and into photosystem I. The same photodepression of O₂ uptake also appears to account for a transient in net O₂ exchange seen in several algae upon turning off the light.     

THE CAROTENOIDS OF FOUR BLUE-GREEN ALGAE1

The carotenoids of 4 species of blue-green algae, Anabaena variabilis, Phormidium persicinum, P. ectocarpi, and P. fragile, were investigated. In each, ft-carotene was a major pigment and the only carotene detected. The xanthophylls present in Anabaena variabilis were echinenone, canthaxanthin, and myxo-xanthophyll. Each of the Phormidium species contained zeaxanthin as the major xanthophyll. In each, this was accompanied by trace amounts of echinenone and isocryptoxanthin. In addition, 2 new xanthophylls, spectrally resembling ^-carotene, were found in Phormidium persicinum and P. ectocarpi, while another, with a spectrum similar to that of myxoxanthophyll, was found in P. fragile.     

Substrate specificities in class A beta-lactamases: preference for penams vs. cephems. The role of residue 237

Site saturation mutagenesis has been carried out at Ala-237 in RTEM-1 beta-lactamase to assess the role of this site in modulating differences in specificity of beta-lactamases for penams vs. cephems as substrates. (An Ala-237 Thr mutation had previously been shown to increase activity on cephems by about 30-80%.) Screening of all 19 possible mutants on penams and cephems revealed the even more active Ala-237 Asn mutant. Detailed kinetic analysis shows that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild-type enzyme. Both mutations reduce the activity toward penams to about 10% that of RTEM-1 beta-lactamase and lower by about 5 degrees C the temperature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most interesting aspect of these results is that two quite disparate amino acids, threonine and asparagine, when introduced for Ala-237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.     

An interdisciplinary approach to the treatment of a hyperfunctional voice disorder

The purpose of this article is to describe the treatment of a 45-year-old male with a hyperfunctional voice disorder by a biofeedback therapist and a speech-language pathologist. The interdisciplinary approach to the treatment of this voice disorder involved the combined use of traditional voice therapy techniques and EMG biofeedback procedures together with cognitive behavioral therapy. Voice therapy was facilitated through the use of a computer-based, speech-monitoring system. The remediation of this voice disorder was attributed to the collaborative efforts of two professionals representing diverse professional training and treatment protocols. The results showed reductions in muscle activity in the infrahyoid and laryngeal areas as well as improved use of proper breathing and voicing onset behaviors. Follow-up at 10 and 15 months posttreatment intervals indicated that the client had retained all target voice skills and the tension reduction/biofeedback skills. Results suggest that interdisciplinary, collaborative efforts using biofeedback and voice therapy can prove beneficial in the treatment of hyperfunctional voice disorders.Note: Neither author has been compensated in any way for the use of the CAFET System nor does either have any financial interest in the company.     

Infectivity of preserved Cryptosporidium parvum oocysts for immunosuppressed adult mice

Abstract The present study was undertaken to determine the infectivity of Cryptosporidium parvum oocysts for immunosup-pressed adult C57BL/6N mice after the oocysts had been stored from 1–48 months at 4°C in 2.5% potassium dichromate. All mice inoculated with oocysts 1–18 months old developed patent infections, while mice inoculated with older oocysts remained uninfected. The prepatent period was extended from 2 to 6 or 7 days as the storage time for oocysts increased. The finding that C. parvum oocysts remain infective for mice for at least 18 months offers important economic and time-saving advantages for investigators who frequently require large numbers of oocysts that must be painstakingly purified from calf manure.     

Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.