Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis

The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomnnose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B withA. saitoi (1–2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

Real-time ultrasonic biparietal diameter measurement for the prediction of gestational age in llamas

Llama (Lama glama ) fetuses (n = 25) of known gestational age (Day 66 to Day 235) were examined transabdominally using real-time ultrasound with a 5 MHz linear-array scanhead. Maximum head width, or biparietal diameter (BPD), was measured in symmetrical images using electronic linear distance calipers. The fetus was easily located and the BPD measured as early as Day 66 of gestation. During the last trimester, measurement became increasingly difficult due to the posture of the fetus and the limited penetration of the 5 MHz frequency. The relationship between days of gestational age (GA) and mean BPD in millimeters is described by the equation: [GA = 18.8 + 3.79 BPD]. Assignment of gestational age allows estimation of parturition date in this frequently pasture-bred and newly polular domestic ruminant.     

Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos

Agrobacterium attached to wheat embryos in vitro. This attachment was plasmid independent, and occurred on both wounded and unwounded cell surfaces. The pattern of attachment clearly demonstrated that bacterial attachment to cereal cells follows the same trends observed for dicotyledonous plants. During the inoculation period the bacterial cells attach to the plant cell walls either with lateral or polar orientation. Wounding (mechanical or enzymatic) preferentially promoted adherence of the bacteria at the wound site, however, attachment was not wound dependent.     

Insulin induces activation and translocation of protein kinase FA (a multifunctional protein phosphatase activator) in human platelet

Protein kinase FA (an activator of the ATP.Mg-dependent multifunctional protein phosphatase) has been identified in both cytosol and plasma membrane isolated from human platelets. The FA activity in the cytosol is active whereas the FA activity in the membrane is inactive. Quantitative analysis further indicates that approximately 90% of total FA is present in the membrane whereas only 10% of FA is localized in the cytosol, suggesting that the inactive membrane-associated FA might be regulated. This notion has subsequently been demonstrated that exposure of platelets to physiological concentrations of insulin for only 1 min resulted in an increase in cytosolic FA activity to about 300% of control values in the absence of insulin and in a corresponding decrease in FA activity in the membrane. It is concluded that the molecular basis for insulin action on cellular metabolism may partly be mediated through the activation and translocation of protein kinase FA in the membrane. It is suggested that redistribution of protein kinase FA may represent a transmembrane signal of insulin.     

Evaluation of a tangential-flow multiple-filter technique for detection of Giardia lamblia cysts in water.

A system of tangential-flow filtration was evaluated for use in the detection of Giardia cysts in drinking water. This method was more sensitive in recovering cysts than a frequently used wound-orlon system of through-filtration.     

The immune response to mouse hepatitis virus: expression of monocyte procoagulant activity and plasminogen activator during infection in vivo

After infection with 10(3) plaque-forming units of mouse hepatitis virus strain 3 (MHV-3) in vivo, peripheral blood mononuclear cells and splenic cells expressed procoagulant activity (PCA) in a pattern directly correlating with susceptibility to disease. Mononuclear cells from BALB/cJ mice, a strain which is fully susceptible to MHV-3, expressed a greater than 500-fold increase in PCA. PCA was first detected within 12 hr of infection; prior to histologic evidence of disease and viral replication, it reached maximal levels 48 hr post-infection (p.i.) and persisted until the death of the animals 5 to 7 days p.i. Mononuclear cells from C3HeB/FeJ mice expressed a significant but lesser titer of PCA, with elevated PCA persisting throughout the chronically infected state until death of the animals 4 to 6 mo p.i. Basal levels of PCA were detected in mononuclear cells from fully resistant A/J mice despite the presence of large amounts of virus in livers, spleens, and sera from these animals. When mononuclear cells expressing high PCA were subfractionated, monocytes were found to be the cellular source of greater than 96% of the PCA activity. Increased plasminogen activator activity was found in monocytes from resistant A/J mice at the time when PCA was markedly elevated in BALB/cJ and C3HeB/FeJ mice. This activity persisted for 5 to 7 days p.i., but was undetectable 10 days p.i. at a time when the mice had cleared the virus from their blood streams. These observations suggest that monocyte PCA may be important in the pathogenesis of MHV-3 disease, whereas the production of monocyte plasminogen activators may contribute to resistance of A/J mice to MHV-3-induced liver disease.     

Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa

Ecdysteroid levels in larvae and pupae of Anastrepha suspensa (Diptera: Tephritidae) were measured by radioimmunoassay. These levels were correlated with histological changes throughout the development of the post-embryonic stages. Ecdysteroid levels increase rapidly throughout the last-larval instar and on the last day of this stage are 283 pg equivalents of 20-hydroxyecdysone per insect (14.5 ng/g) when wandering behaviour is initiated. At the end of this period the puparium is formed and within 24 h, the ecdysteroid rises to its highest peak (625 pg equivalents of 20-hydroxyecdysone/insect). Larval-pupal apolysis is initiated within 24 h later and the pupal cuticle is then secreted. Two days later, the ecdysteroids reach their lowest levels (75 pg equivalents of 20-hydroxyecdysone/insect or 0.6 ng/g) and most of the larval fat body and other tissues have been histolysed. In 5- to 10-day old pupae ecdysteroid levels again increase and remain relatively high throughout. During this period the larval epidermis is replaced by imaginal epidermis, imaginal discs begin to proliferate and the adult cuticle is secreted. Ecdysteroid levels finally fall 2 days prior to adult emergence. HPLC determinations indicate that 20-hydroxyecdysone is the predominant free ecdysteroid, and along with ecdysone, is readily detectable in all postembryonic stages of this species. An unusually high and unexplained peak of 20-hydroxyecdysone occurs in the pharate adult. This peak probably consists of ecdysone metabolites with retentions similar to that of 20-hydroxyecdysone and to which the antiserum is sensitive.