Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD₅₀ at 28 days post infection ranged from 3.6–7.5×10⁵ cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.     

Pachytene analysis in males heterozygous for a familial translocation (9;12;13) (q22; q22; q32) ascertained through a child with partial trisomy 9

A family with four male and three female heterozygotes for a three-way translocation (9;12;13) (q22; q22; q32) in three generations was ascertained through a chromosomally imbalanced newborn with an additional derivative chromosome 9 resulting from nondisjunction. Three heterozygous males from two generations with apparent differences in their fertility status were investigated using pachytene spreads and testicular histology. Pachytene analysis in all three individuals, the fertile (II-2) as well as the subfertile (III-7) and infertile (III-9), showed a hexavalent with central nonpairing around the translocation breakpoints in nearly all spermatocytes. Thus, the observed hexavalent configurations in pachytene do not seem to have caused impaired fertility. This rather may have been the result of sperm carrying unbalanced chromosome sets. However, the observed difference in fertility between the heterozygous fertile male in generation II and his two heterozygous sons remains unexplained.     

Neonatal screening for congenital adrenal hyperplasia using 17-hydroxyprogesterone assay in filter paper blood spots

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

Anisotropy decay of fluorescence as an experimental approach to protein dynamics

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.     

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.     

Biochemical correlates of fatigue. A brief review

Muscle fatigue, defined as a decreased force generating capacity, develops gradually during exercise and is distinct from exhaustion, which occurs when the required force or exercise intensity can no longer be maintained. We have reviewed several biochemical and ionic changes reported to occur in exercising muscle, and analysed the possible effects these changes may have on the electrical and contractile properties of the muscle. There is no evidence that substrate depletion can account for the decreased force generating capacity, but this factor may be important for the rate of energy turnover and be a major determinant for endurance. Increased concentration of inorganic phosphate and hydrogen ions will depress the force generating capacity, but since fatigue can develop gradually without accumulation of these ions they can only be important when aerobic ATP production is insufficient to support the contractions. Evidence is presented showing that a disturbed balance of K+ alone might cause depolarisation block at high stimulation frequencies, but extracellular K+ accumulation does not increase gradually during prolonged dynamic or static exercise, and is therefore not closely related to fatigue. The repeated release of Ca2+ from the sarcoplasmic reticulum (SR) during muscular activity is suggested of Ca2+ by the mitochondria, increasing with stimulation frequency and duration and possibly also deteriorating mitochondrial function. We therefore speculate that decreased Ca2+ availability for release from SR might contribute to a gradual decline in force generating capacity during all types of exercise.     

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).