In vitro reconstitution of the tracheal epithelium.

We have developed a unique in vitro reconstitution system for tracheal epithelia of guinea pigs. In the system, a human amnion membrane was used as a basement membrane and the tracheal epithelial cells were cultured on the epithelial side of the membrane. Three weeks later, the tracheal fibroblasts were co-cultured on the serosal side of the amnion membrane and the culturing was continued for an additional 10 d. The morphology of the cultured epithelial cells consisted of a pseudostratified columnar ciliated epithelium from cuboidal ciliated epithelium during the last 10 d of the culture period. Epithelial cells included both goblet-like and basal cells. In addition, the frequency of each type of differentiated cells was almost identical to that of in vivo tracheas. Interestingly, the same results were obtained when the conditioned medium of the tracheal fibroblasts was used instead of the fibroblasts themselves. These results suggest that epithelial-mesenchymal interaction is likely involved in growth and differentiation of epithelial cells in vivo in a soluble factor(s)-mediated manner. As well as the epithelial cells, the fibroblasts also formed a multilayer during the last 10 d of co-culturing. This indicates that in vitro reconstitution of tracheal epithelia is achieved without addition of any exogenous growth or differentiation factors. The reconstitution system is shown to be useful for investigating the cellular and molecular interaction of epithelial and mesenchymal cells. Possible applications of the culture system and possible factors involved in growth and differentiation of epithelial cells are discussed.

An exploration of family-centred care in Neuman's model with regard to the care of the critically ill adult in an accident and emergency setting.

In Accident and Emergency (A & E) departments there is a tendency to separate relatives from critically ill patients, and although in many departments the relatives have a nurse allocated to care for them, this care is often given in a separate 'relatives' room. This article explores the meanings of family, family nursing and family systems nursing in the context of an A & E setting and using Neuman's (1989) model to highlight family dynamics. The intention is to allow the thinking A & E nurse to reflect on care given in the department, so that individualised care can be given with consideration to the relationships that the person has before they take on the status of 'patient'. Thus, the article is meant to be thought-provoking rather than prescriptive.

Coordinating locally 'owned' treatment guidelines.

South West Thames Regional Health Authority established and commissioned a regional guidelines unit to coordinate the introduction of a set of treatment guidelines on the management of common medical emergencies into all the acute intaking National Health Service (NHS) hospitals throughout the region. All hospitals were offered a set of template guidelines to be used at their discretion for producing their own customised equivalent. They were also offered full typing and production facilities, together with printing costs if publication was achieved by a target deadline (1 August 1993). In 11 of the 14 NHS hospitals guidelines were available to hospital staff by the target deadline, and one set was produced for a non-NHS hospital. In two hospitals the target date was not met, and one other declined to take part. As part of the project the unit assessed the extent to which the published guidelines were adapted to meet the requirements of each individual hospital. The template offered guidelines on 34 topic titles. No hospital used all core titles of the original template; titles were omitted or replaced in some, and added in others. Where the original guideline titles were used, there was almost always some customisation--changes in sentence structure, names or contact numbers, alterations in drugs and doses or the addition or omission of entire sections. By using an established resource, sets of customised, locally determined treatment guidelines were introduced with relative ease into most of the acute hospitals in a UK health region.

Fetal growth achievement and elevated maternal serum alpha-fetoprotein.

Maternal serum alpha-fetoprotein (AFP) was measured by radioimmunoassay in 7223 unselected patients between 16 and 20 weeks gestation. In 141 patients an elevated AFP level (greater than 2.5 multiples of the median for gestation) was found in the absence of a primary cause. When the birthweights of the 137 liveborn infants were corrected for maternal height and weight, sex and birth rank, 37 (27%) fell below the 10th centile of normal birthweight standards. No excess of premature deliveries was found, but there was a significant association with primiparity. Patients delivered of their second infant showed a significant decrement in mean birthweight when compared with their first-born infants and with a matched control group (normal maternal serum AFP levels). There was a highly significant association between elevated serum AFP and subsequent placental abruption.

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3.

Glycogen synthase kinase-5 (casein kinase-II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C-terminal to the 3 serines phosphorylated by glycogen synthase kinase-3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase-3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase-5 would appear to be a novel one in forming the recognition site for another protein kinase.

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain.

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].

The regulation of muscle phosphorylase kinase by calcium ions, calmodulin and troponin-C.

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase in vivo is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism of coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the delta-subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state. Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45-49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions.

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.

Phosphorylase kinase from rabbit skeletal muscle: identification of the calmodulin-binding subunits.

Phosphorylase kinase has the structure (alpha beta gamma delta)4 where the delta-subunit is identical to the calcium-binding protein termed calmodulin [Shenolikar et al. (1979) Eur. J. Biochem. 100, 329--337]. The delta-subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The delta-subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the delta-subunit was associated with the gamma-subunit. This finding was confirmed by cross-linking experiments with dimethylsuberimidate which resulted in the formation of a gamma delta complex. Phosphorylase kinase was shown to bind one additional molecule of calmodulin per alpha beta gamma delta unit, termed the delta'-subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the delta'-subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 microM. This was similar to the concentration of delta'-subunit which produced half-maximal activation. The delta'-subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the delta-subunit and [14C]calmodulin, and the calcium-dependent binding of the delta'-subunit allowed cross-linking experiments to be used which demonstrated that the delta'-subunit was bound to both the alpha and beta subunits. This result was supported by the finding that selective proteolysis of either the alpha-subunit, or the alpha and beta subunits, decreased or abolished the ability of phosphorylase kinase to bind to calmodulin-Sepharose. The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.

Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin.

Although it has been believed for several years that Ca2+ are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past two years that this concept has started to be placed on a firm molecular basis. The current evidence suggests that the regulation of phosphorylase kinase by Ca2+ in vivo is achieved through the interaction of this divalent cation with calmodulin (the delta subunit) and troponin C, and that the relative importance of these two calcium binding proteins depends on the state of phosphorylation of the enzyme [FIGURE 1]. In the low-activity dephosphorylated b form, increasing Ca2+ from 0.1 microM to concentrations in the microM range produces a 5-10-fold activation through the binding of Ca2+ to the delta subunit, and a further 15-25-fold activation through the binding of Ca2+ to troponin C [TABLE 1]. Troponin C rather than the delta subunit is therefore the dominant calcium dependent regulator of the b form, providing an attractive mechanism for coupling glycogenolysis and muscle contraction. On the other hand, the high-activity phosphorylated a form is only activated very slightly by troponin [Section 7]. The delta subunit is therefore the dominant calcium dependent regulator of the hormonally activated state of the enzyme. It has recently become clear that phosphorylase kinase not only phosphorylates and activates phosphorylase, but also phosphorylates glycogen synthase, decreasing its activity. The regulation of phosphorylase kinase by Ca2+ may therefore also provide a mechanism for achieving synchronous control of the pathways of glycogenolysis and glycogen synthesis.

The conversion of 3-Hydroxy-2,4,5-trihydroxymethylpyridine into pyridoxine by Kloeckera apiculata.

Kloeckera apiculata, a vitamin B-6-dependent yeast, grows in the presence of 3-hydroxy-2,4,5-trihydroxymethylpyridine in vitamin B-6-free media. On a molar basis the growth-promoting activity of this compound is approximately one-tenth that of other forms of vitamin B-6. [G-3H]3-Hydroxy-2,4,5-trihydroxymethylpyridine is converted into radioactive vitamin B-6 compounds of the same specific radioactivity by growing cultures of K. apiculata.