Decisive role of intermediate filament typing of tumor cells in the differential diagnosis of difficult fine needle aspirates

Thirty-six diagnostically difficult fine needle aspirates from enlarged lymph nodes and malignant soft tissue tumors, containing tumor cells with scanty or no obvious light microscopic features indicative of their differentiation, were assessed by a panel of six cytopathologists. Their diagnoses were recorded and then compared with the definitive diagnosis established by combining the cytologic findings with the results of intermediate filament typing of tumor cells in the smears using monoclonal antibodies specific for each filament type. The results show that use of these antibodies can markedly improve the accuracy of the cytologic diagnosis of tumor type as well as revise or prevent erroneous cytologic diagnoses in difficult cases. This pertains especially to the differential diagnoses of carcinoma versus malignant lymphoma, carcinoma versus malignant melanoma, carcinoma versus sarcoma and squamous carcinoma versus carcinoma of simple epithelia. Intermediate filament typing of tumor cells in aspirates as an objective histogenetic criterium makes the differential diagnosis of the difficult aspirates much more reliable and reproducible, provided that appropriate questions are asked, monoclonal antibodies with well-defined specificities are used and the antigenicity of the intermediate filaments in smears is preserved.     

Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.     

Reflex control of inspiratory duration in newborn infants

We applied graded resistive and elastic loads and total airway occlusions to single inspirations in six full-term healthy infants on days 2-3 of life to investigate the effect on neural and mechanical inspiratory duration (TI). The infants breathed through a face mask and pneumotachograph, and flow, volume, airway pressure, and diaphragm electromyogram (EMG) were recorded. Loads were applied to the inspiratory outlet of a two-way respiratory valve using a manifold system. Application of all loads resulted in inspired volumes decreased from control (P less than 0.001), and changes were progressive with increasing loads. TI measured from the pattern of the diaphragm EMG (TIEMG) was prolonged from control by application of all elastic and resistive loads and by total airway occlusions, resulting in a single curvilinear relationship between inspired volume and TIEMG that was independent of inspired volume trajectory. In contrast, when TI was measured from the pattern of airflow, the effect of loading on the mechanical time constant of the respiratory system resulted in different inspired volume-TI relationships for elastic and resistive loads. Mechanical and neural inspired volume and duration of the following unloaded inspiration were unchanged from control values. These findings indicate that neural inspiratory timing in infants depends on magnitude of phasic volume change during inspiration. They are consistent with the hypothesis that termination of inspiration is accomplished by an "off-switch" mechanism and that inspired volume determines the level of vagally mediated inspiratory inhibition to trigger this mechanism.     

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.     

Construction and use of a nontoxigenic strain of Pseudomonas aeruginosa for the production of recombinant exotoxin A.

To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.     

Sexual dimorphism in the developmental regulation of brain aromatase

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase (cytochrome P450_AR), converting testosterone (T) to estradiol-17β (E₂) is a key enzyme in brain development and the regulation of aromatase determines the availability of E₂ effective for neural differentiation. Gender differences in brain development and behaviour are likely to be influenced by E₂ acting during sensitive periods. This differentiating action has been demonstrated in rodent and avian species, but also probably occurs in primates including humans. In rodents, E₂ is formed in various hypothalamic areas of the brain during fetal and postnatal development. The question considered here is whether hypothalamic aromatase activity is gender-specific during sensitive phases of behavioural and brain development, and when these sensitive phases occur. In vitro preoptic and limbic aromatase activity has been measured in two strains of wild mice, genetically selected for behavioural aggression based on attack latency, and in the BALB/c mouse. Short attack latency males show a different developmental pattern of aromatase activity in hypothalamus and amygdala to long attack latency males. Using primary brain cell cultures of the BALB/c mouse, sex differences in hypothalamic aromatase activity during both early embryonic and later perinatal development can be demonstrated, with higher E₂ formation in males. The sex dimorphisms are brain region specific, since no differences between male and female are detectable in cultured cortical cells. Immunoreactive staining with a polyclonal aromatase antibody identifies a neuronal rather than an astroglial localization of the enzyme. T increases fetal brain aromatase activity and numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies. T appears to influence the growth of hypothalamic neurons containing aromatase. Differentiation of sexually dimorphic brain mechanisms may involve maturation of a gender-specific network of estrogen-forming neurons which are steroid-sensitive in early development.     

Focal adhesion regulation of cell behavior

Focal adhesions lie at the convergence of integrin adhesion, signaling and the actin cytoskeleton. Cells modify focal adhesions in response to changes in the molecular composition, two-dimensional (2D) vs. three-dimensional (3D) structure, and physical forces present in their extracellular matrix environment. We consider here how cells use focal adhesions to regulate signaling complexes and integrin function. Furthermore, we examine how this regulation controls complex cellular behaviors in response to matrices of diverse physical and biochemical properties. One event regulated by the physical structure of the ECM is phosphorylation of focal adhesion kinase (FAK) at Y397, which couples FAK to several signaling pathways that regulate cell proliferation, survival, migration, and invasion.     

Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.