Adeno-associated virus rep protein synthesis during productive infection.

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. We studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with [35S]methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.     

Blood flow to the placenta and lower body in the growth-retarded guinea pig fetus

Blood flow to the placenta and lower body of control and growth retarded (IUGR) guinea pig fetuses was measured between 60-64 days of pregnancy by the microsphere technique. Further information about the hepatic blood supply and its interlobular distribution was obtained by injecting microspheres into the umbilical vein and a branch of the portal vein. Liver weight was reduced by 60% in IUGR fetuses from 5.0 +/- 0.2 to 2.0 +/- 0.1 g, compared to a decrease in body weight of 50% from 91.6 +/- 3.0 to 45.4 +/- 2.6 g. In addition, there was a proportionately greater reduction in the size of the right liver lobe. Umbilical blood flow was 10.8 +/- 1.0 ml min-1 in control fetuses and 4.9 +/- 1.2 ml.min-1 in IUGR fetuses, whilst blood flow in the portal vein was reduced from 1.4 +/- 0.1 to 0.8 +/- 0.3 ml min-1 and that in the hepatic artery from 0.6 +/- 0.1 to 0.3 +/- 0.1 ml.min-1. Since ductus venosus flow was absent or negligible, the umbilical venous return accounted for greater than 80% of the hepatic blood supply in both control and IUGR fetuses. Blood flows were, however, unequally distributed between the liver lobes. The right lobe was supplied mainly by the portal vein in IUGR fetuses as well as the controls, and received less than 6% of the umbilical venous return. No significant change occurred in total liver perfusion, which was 2.8 +/- 0.2 ml min-1 per g in control fetuses and 2.6 +/- 0.4 ml min-1 per g in IUGR fetuses. It is therefore suggested that a high rate of liver metabolism is maintained in IUGR, but by a smaller tissue mass, and that the rate of umbilical blood flow may be one factor determining the size of the liver. The relatively greater reduction in size of the right lobe in IUGR is probably the result of poor oxygenation of the portal venous blood.     

DEVELOPMENTAL STABILITY IN LEAVES OF CLARKIA TEMBLORIENSIS (ONAGRACEAE) AS RELATED TO POPULATION OUTCROSSING RATES AND HETEROZYGOSITY

Four natural populations of Clarkia tembloriensis, whose levels of heterozygosity and rates of outcrossing were previously found to be correlated, are examined for developmental instability in their leaves. From the northern end of the species range, we compare a predominantly selfing population (t? = 0.26) with a more outcrossed population (t? = 0.84), which is genetically similar. From the southern end of the range, we compare a highly selfing population (t? = 0.03) with a more outcrossed population (t? = 0.58). We measured developmental stability in the populations using two measures of within-plant variation in leaf length as well as calculations of fluctuating asymmetry (FA) for several leaf traits. Growth-chamber experiments show that selfing populations are significantly more variable in leaf length than more outcrossed populations. Developmental instability can contribute to this difference in population-level variance. Plants from more homozygous populations tend to have greater within-plant variance over developmentally comparable nodes than plants from more heterozygous populations, but the difference is not significant. At the upper nodes of the plant, mature leaf length declines steadily with plant age, allowing for a regression of leaf length on node. On average, the plants from more homozygous populations showed higher variance about the regression (MSE) and lower R² values, suggesting that the decline in leaf length with plant age is less stable in plants from selfing populations than in plants from outcrossing populations. Fluctuating asymmetry (FA) was calculated for four traits within single leaves at up to five nodes per plant. At the early nodes of the plant where leaf arrangement is opposite, FA was also calculated for the same traits between opposite leaves at a node. Fluctuating asymmetry is significantly greater in the southern selfing population than in the neighboring outcrossed population. Northern populations do not differ in FA. Fluctuating asymmetry can vary significantly between nodes. The FA values of different leaf traits were not correlated. We show that developmental stability can be measured in plants using FA and within-plant variance. Our data suggest that large differences in breeding system are associated with differences in stability, with more inbred populations being the least stable.     

Disruption of CENP antigen function perturbs dynein anchoring to the mitotic kinetochore

Injection of purified autoantibodies against human centromeric proteins into HeLa cells during interphase disrupts the organization of the kinetochore and interferes with chromosomal movements during the subsequent mitosis even though the chromosomes retain the ability to bind microtubules. We have investigated the hypothesis that this phenotype arises from effects on cytoplasmic dynein, the microtubule motor protein. In previous experiments we found that introduction of anticentromere antibodies into cell nuclei during the G₁- or S-phases causes a prometaphase-like arrest, while injections during G₂-phase cause a metaphase arrest. We show here that, in both cases, the level of detectable cytoplasmic dynein at kinetochores is significantly decreased. In contrast, when injected cells were permitted to enter mitosis in the absence of microtubules (conditions where trilaminar kinetochores could be detected by electron microscopy), the intensity of dynein labeling on the kinetochores was identical to that seen in uninjected control cells exposed to colcemid. Therefore, the loss of dynein label on mitotic kinetochores was correlated both with the injection of anticentromere antibodies and with the presence of intact spindle microtubules. We suggest that the injection of anticentromere antibodies somehow weakens the association of dynein with the kinetochore, so that when microtubules are present, these motor molecules are pulled away from the kinetochores as they generate force. This model offers an explanation for the failure of chromosomes of injected cells to move normally in mitosis even though they have attached microtubules.     

Perinatal Steroid Treatments Alter Alloparental and Affiliative Behavior in Prairie Voles

This experiment was designed to examine the hypothesis that perinatal manipulation of gonadal or adrenal steroids can alter the subsequent expression of juvenile parental (alloparenting) and affiliative behavior in prairie voles (Microtus ochrogaster). Corticosterone (PRECORT), testosterone (PRE-TP), or oil injections (PRESES) were given on Prenatal Days 12–20 or on Postnatal Days 1–6 (CORT6, TP6, or SES6, respectively). Alloparenting was reduced significantly in females in the CORT6 group and in males in the TP6 group. Sibling affiliative preferences were increased significantly in PRE-TP females and stranger preferences were increased in TP6 and CORT6 females. The results suggest timing is a critical factor determining whether hormones have a facilitative or inhibitory effect on alloparental and affiliative behavior in prairie voles. In this species, corticosterone and testosterone have similar organizational effects on affiliative behavior in females. Alloparental behavior is inhibited by postnatal corticosterone administration in females and by postnatal testosterone administration in males, whereas prenatal steroid administration had no significant effect on alloparenting in either gender.     

The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.

A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

A framework for the analysis of gender,intra-household dynamics,and livestock disease control with examples from Uasin Gishu district,Kenya

As livestock disease control programs in Africa begin to rely more upon para-professionals and livestock producers as deliverers of animal health care services, understanding the role different household members play in providing animal health care becomes increasingly important. This paper presents a framework for the analysis of gender aspects of livestock disease control based on a similar framework developed by Feldstein and Poats (1989). The utility of this framework is illustrated using household-level data collected from a district in central Kenya. Adult women and elderly men in the sample have primary responsibility for livestock care, and are therefore well placed to diagnose illness. Dipping and spraying of animals to prevent tick-borne and other diseases is the primary responsibility of adult males. Decisions regarding use of milk from the morning milking are more likely to be made by adult men. It is morning milk that is most often sold. Adult women, however, make decisions about use of evening milk, which is most often kept for household consumption. Knowledge of livestock diseases did not appear to vary significantly by gender, although some elderly men did possess extensive knowledge of indigenous disease categories and traditional remedies. The importance of recognizing gender issues in planning and implementing livestock disease control programs is discussed.