Use of the granulosa cell aromatase bioassay for measurement of bioactive follicle-stimulating hormone in urine and serum samples of diverse species

Ovarian steroids and growth factors are intragonadal modulators which augment a key endpoint of follicle-stimulating hormone (FSH) action in granulosa cells: the induction of aromatase activity. Studies of these paracrine hormones that enhance FSH-stimulated estrogen biosynthesis by cultured rat granulosa cells, have led to the development of a sensitive and specific bioassay for FSH. This newly developed granulosa cell aromatase bioassay (GAB) allows for the measurement of bioactive FSH levels in serum and urine of humans and animals with various physiological and pathological conditions. These studies have demonstrated that the GAB assay is useful in detecting possible changes in the molecular forms of FSH. The adaptation of this method for urine samples allows for the measurement of bio-FSH levels in situations where venipuncture is not practical or in species for which specific radioimmunoassays are not available.     

C-band polymorphisms of chromosome 9: quantification by Ce-bands

Summary C-band polymorphisms of chromosome 1 can be quantified by C_e bands visualized using oblique epi-illumination. In this paper the polymorphic region of chromosome 9, including variants such as 9qh+, inversions, and translocations, was analyzed in a total of 1860 chromosomes from 20 individuals and 8 fetuses. In this sample we found between zero and five C_e bands on 9q and between zero and four C_e bands on 9p with a maximum of five C_e bands per chromosome. On the basis of these observations at least 19 different polymorphic patterns can be expected theoretically, 15 of which were observed in our sample. Among these, five polymorphic C_e-band classes were distinguishable by the method presented. Considering both homologues, 5²=25 quantitatively discernible chromosome 9 pairs may exist.     

Social context and reaction to separation in peer-reared pigtail macaques: Some preliminary observations

The social environment affects both behavioral and physiological responses to separation from the mother. Less information is available on the impact of the social environment on the response to separation in peer-reared infant monkeys. This study reports the responses of peer-reared pigtail macaque infants to repeated separations, and the impact of social versus isolation housing during the separation. The responses of two pairs of monkeys were studied during four three-day separations. One of each pair was housed in isolation during the separation, and the other was with another pair of peers, with whom they had been living for one month prior to the separation. The isolation-housed peer responded to the separation with behavioral agitation, but no depression. The socially-housed peer's behavior did not differ from baseline during the separation. During successive reunions, all the separated monkeys, regardless of housing condition, exhibited declining levels of behaviors related to maintaining proximity to their attachment figure. Although the number of subjects is small, the results suggest that the presence of social support, in the form of a familiar peer, can ameliorate the response to separation, and that with repeated separations the responses of the monkeys changes significantly.     

Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27.

Infected-cell protein 27 (ICP27) is a herpes simplex virus type 1 alpha, or immediate-early, protein involved in the regulation of viral gene expression. To better understand the function(s) of ICP27 in infected cells, we have isolated and characterized viral recombinants containing defined alterations in the ICP27 gene. The mutant virus d27-1 contains a 1.6-kilobase deletion which removes the ICP27 gene promoter and most of the coding sequences, while n59R, n263R, n406R, and n504R are mutants containing nonsense mutations which encode ICP27 molecules truncated at their carboxyl termini. All five mutants were defective for lytic replication in Vero cells. Analysis of the mutant phenotypes suggests that ICP27 has the following regulatory effects during the viral infection: (i) stimulation of expression of gamma-1 genes, (ii) induction of expression of gamma-2 genes, (iii) down regulation of expression of alpha and beta genes late in infection, and (iv) stimulation of viral DNA replication. Cells infected with the mutant n504R expressed wild-type levels of gamma-1 proteins but appeared to be unable to efficiently express gamma-2 mRNAs or proteins. This result suggests that ICP27 mediates two distinct transactivation functions, one which stimulates gamma-1 gene expression and a second one required for gamma-2 gene induction. Analysis of the mutant n406R suggested that a truncated ICP27 polypeptide can interfere with the expression of many viral beta genes. Our results demonstrate that ICP27 has a variety of positive and negative effects on the expression of viral genes during infection.     

Restricted expression of herpes simplex virus lytic genes during establishment of latent infection by thymidine kinase-negative mutant viruses.

Infection of cells by herpes simplex virus (HSV) can lead to either lytic, productive infection or nonlytic, latent infection. The factors influencing this infection pathway decision are largely unknown. Thymidine kinase-negative mutant viruses can establish latent infection in neurons of mouse trigeminal ganglia but do not replicate productively in these cells. We show that during the early stages of establishment of latency by these mutants, expression of viral lytic genes is drastically reduced or undetectable as assayed by in situ hybridization. Thus, establishment of latent infection by HSV can occur despite severely restricted levels of lytic gene expression. This suggests that the block to productive replication during establishment of latent infection by HSV occurs before or early during the expression of alpha genes.     

The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves

Shoot regeneration from Rubus leaves was obtained on a medium containing MS salts, vitamins and sugars, Staba vitamins, casein hydrolysate (100 mg l^–1) and 10 M thidiazuron. Shoot regeneration from Malus leaves was obtained on N⁶ rice anther medium with 5 M thidiazuron. In vitro pretreatment of source shoots with either colchicine or thidiazuron enhanced the organogenic potential of detached leaves of two Rubus hybrids. The response to colchicine was quadratic and occurred at non-mutagenic concentrations (75–250 M). The response to thidiazuron was exponential between 0 and 5 M. When applied as a pretreatment, the effectiveness of several different cytokinins (benzyladenine, thidiazuron, zeatin) at enhancing Malus and Rubus organogenesis was related to the shoot proliferation activity of the cytokinin and to treatment-induced variation in leaf and petiole size.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - MS Murashige & Skoog basal medium devoid of plant growth regulators - OI organogenesis-initiating subculture - PTI colchicine pretreatment subculture - PTII cytokinin pretreatment subculture - NAA naphthaleneacetic acid - TDZ thidiazuron - zeatin trans-zeatin     

Viral replication is required for induction of ocular immunopathology by herpes simplex virus.

Corneal infection of BALB/c mice with herpes simplex virus type 1 results in a chronic inflammatory response in the stroma termed herpetic stromal keratitis (HSK). This disease is considered to be immunopathological and mediated primarily by CD4+ T cells of the type 1 cytokine profile. However, the nature of the antigens, virus or host derived, which drive the inflammatory response remains in doubt. In this study, the relevance of infection with replicating virus for the subsequent development of HSK was evaluated with immunocompetent mice as well as with SCID mice reconstituted with herpes simplex virus-immune CD4+ T cells. In the corneas of immunocompetent mice, infectious virus, viral antigen, and mRNA expression were detectable for only a brief period of time (< or = 7 days postinfection), and all were undetectable by the time clinical lesions were evident (10 to 15 days). Viral replication, however, was necessary for the development of HSK in both models, since infection with UV-inactivated virus or with mutant viruses which were incapable of multiple rounds of replication in vivo failed to induce HSK. The inactivated and mutant viral preparations did, however, stimulate T-cell immune responses in immunocompetent mice. The results are discussed in terms of possible involvement of host antigens exposed in response to transient progeny virion replication in the immune-privileged cornea.     

Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication.

Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule.     

The direction of mothers' and daughters' preferences and the heritability of male ornaments in red jungle fowl (Gallus gallus)

We used a breeding design involving 18 sires and 108 dams tostudy the heritabilities of male ornaments in red jungle fowl(Gallus gallus). Ornaments used by females to choose mates showedlow heritabilities, with the exception of comb and wattle measures.The general absence of heritability suggests that a geneticcovariance did not exist at the time of this study between mostmale ornaments and female preferences for those ornaments. Thisresult is contrary to a key prediction of the arbitrary or Fisherianhypothesis of sexual selection. Comb size and color are condition-dependenttraits that reflect short-term changes in health, and comb sizeof males was positively correlated with offspring weight. Ourresults are consistent with the expectation of good-genes hypothesesthat male ornaments reflect the ability of males to withstandenvironmental stresses.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .