Phenotype of adult Refsum disease due to a defect in peroxin 7.

The biochemical hallmark of adult Refsum disease (ARD) is an isolated deficiency in the breakdown of phytanic acid. This usually results from a PHYH gene defect, although some cases have been found to carry a PEX7 defect. We describe the phenotype of such a patient, indistinguishable from that of classic ARD. Hence, we propose the subdivision of ARD into type 1 and type 2, depending on which gene is defective.

Modified autogenous latissimus breast reconstruction and the box top nipple.

During the past 3 years, the authors have been using the modified autogenous latissimus myocutaneous flap (MALF) for breast reconstruction in increasing numbers because of occasional patient and surgeon dissatisfaction with other methods of breast reconstruction. They have found this method to have unprecedented reliability, making it preferable to other forms of reconstruction in many patients. Considering the very low morbidity, the high patient satisfaction, and current economic factors, the authors are strong advocates of this form of reconstruction. A procedural outline proposed by McCraw and coworkers is followed, with some useful modifications. An elliptical transverse skin paddle is centered over the back fat roll. The area of the skin ellipse measures approximately 8 +/- 2 cm vertically and 30 +/- 5 cm transversely. After making the skin incision, a feathering technique is used in all directions through the fatty layer overlying the latissimus and in the tissue beyond the anteroposterior borders of the latissimus (not beyond 5 cm from the skin incision). By means of feathering, the shape of a breast mound can be created in the allowable tissue supported by the latissimus. A 180-degree rotation of the flap allows dependentvenous drainage and more bulk in the inferior outer quadrant, where it is needed. In the current series of 47 modified autogenous latissimus breast reconstructions, seromas were common. Other complications included one wound infection, one ulnar neuropraxia, and one fat necrosis. There were no flap necroses (partial or complete) or hematomas. The rarity of complications supports the use of this technique in selected patients. An innovative new technique for nipple reconstruction is also described. The "box top technique" of nipple reconstruction consists of four deepithelialized local flaps covered with a skin graft from the groin.

Occupational aspirations of black South African adolescents.

The present study provides a description of the occupational aspirations of 216 black high school students in a special program by the amount of training required (status) and Holland's 1973 typology as well as by gender, age, socioeconomic status, knowledge of self, and occupational knowledge. Analysis indicates that most adolescents aspire to Social and Investigative occupations, and occupations with a high status. Most of this select sample displayed low self- and occupational knowledge. Aspirations appear unrealistic in terms of trends within the labor market, but might be more realistic with effective and relevant guidance programs in schools.

Interaction of a protein phosphatase with an Arabidopsis serine-threonine receptor kinase.

A protein phosphatase was cloned that interacts with a serine-threonine receptor-like kinase, RLK5, from Arabidopsis thaliana. The phosphatase, designated KAPP (kinase-associated protein phosphatase), is composed of three domains: an amino-terminal signal anchor, a kinase interaction (KI) domain, and a type 2C protein phosphatase catalytic region. Association of RLK5 with the KI domain is dependent on phosphorylation of RLK5 and can be abolished by dephosphorylation. KAPP may function as a signaling component in a pathway involving RLK5.

Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana.

The RLK5 gene of Arabidopsis thaliana encodes a novel receptor-like protein kinase. DNA sequence analysis suggests that the RLK5 protein contains an extracellular domain that has 21 tandemly repeated leucine-rich motifs linked, via a transmembrane hydrophobic region, to a protein kinase catalytic domain that is related to the serine/threonine family of protein kinases. To study the intrinsic biochemical properties of this protein kinase we have expressed the catalytic domain as two different recombinant fusion proteins in Escherichia coli. Both hybrid proteins have similar kinetic properties, autophosphorylate on serine and threonine residues and have significantly greater activity in the presence of Mn2+ than Mg2+. A lysine to glutamic acid substitution in the catalytic domain of RLK5 results in the catalytically inactive protein RLK5(Cat)K711E. The active RLK5 protein can phosphorylate the inactive K711E protein and the K711E protein can partially inhibit the autophosphorylation of RLK5. Tryptic cleavage of the autophosphorylated proteins followed by two-dimensional thin layer electrophoresis indicates that several sites in the catalytic domain are phosphorylated.

Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products.

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Klebsiella type 3 fimbriae (pFK10) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteria was inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonal immunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to the basal epithelial cells and the basement membrane regions of bronchial epithelia. Using an E. coli transformant (pDC17/pFK52), which expresses nonadherent P fimbrial filaments, along with the type 3 fimbrial adhesin (MrkD), we demonstrated that type 3 fimbrial attachment to respiratory cells was attributable to the MrkD adhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesin was most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, in vitro, the Klebsiella type 3 fimbrial adhesin mediates adherence to human respiratory tissue.

Characterization of parameters influencing receptor-mediated endocytosis in cultured soybean cells.

In a recent publication, we were able to demonstrate that biotin enters plant cells by receptor-mediated endocytosis and that impermeable macromolecules can be cotransported into cells by the same pathway if they are first covalently linked to biotin. In the present study, we have exploited the biotin endocytosis pathway to evaluate the variables in the cell wall and surrounding growth medium that influence the efficiency of endocytosis in plants. Under normal growth conditions, the major constraint limiting macromolecule endocytosis was found to be the size of the internalized macromolecule. Thus, a log-linear relationship with a negative slope exists between the molecular weight of the biotin-conjugated macromolecule and its rate of internalization by cultured soybean cells. This relationship, which extends from insulin (M(r) approximately 5700) to immunoglobulin G (M(r) approximately 160,000), is characterized by a slope of -1.04 x 10(5) molecules/cell/min per log M(r) unit and an x intercept (no endocytosis detectable) of approximately log 160,000 daltons. Unfortunately, mild digestion with cell wall-degrading enzymes is unable to increase significantly the upper size limit of molecules that can be internalized, but uptake of lower molecular weight proteins can be enhanced by mild cell wall digestion. The optimal extracellular pH for endocytosis was found to be 4.6, i.e. near the normal pH of the cell culture medium. Furthermore, the osmotic strength at which endocytosis occurs most rapidly was observed to be isotonic to slightly hypotonic, suggesting that turgor pressure within the plant cell must not be a major determinant of endocytosis rates by cultured soybean (Glycine max) cells. Finally, cell age was found to impact significantly on the rate of macromolecule internalization, with maximal uptake rates occurring during early exponential growth and decreasing by a factor of 2 when the cells reach stationary growth phase.

Effect of Elicitation and Changes in Extracellular pH on the Cytoplasmic and Vacuolar pH of Suspension-Cultured Soybean Cells.

We have employed both (31)P nuclear magnetic resonance spectroscopy and two intracellular fluorescent pH indicator dyes to monitor the pH of the vacuole and cytoplasm of suspension-cultured soybean cells (Glycine max Merr cv Kent). For the (31)P nuclear magnetic resonance studies, a flow cell was constructed that allowed perfusion of the cells in oxygenated growth medium throughout the experiment. When the perfusion medium was transiently adjusted to a pH higher than that of the ambient growth medium, a rapid elevation of vacuolar pH was observed followed by a slow (approximately 30 minute) return to near resting pH. In contrast, the concurrent pH changes in the cytoplasm were usually fourfold smaller. These data indicate that extracellular pH changes are rapidly communicated to the vacuole in soybean cells without significantly perturbing cytoplasmic pH. When elicitors were dissolved in a medium of altered pH and introduced into the cell suspension, the pH of the vacuole, as above, quickly reflected the pH of the added elicitor solution. In contrast, when the pH of either a polygalacturonic acid or Verticillium dahliae elicitor preparation was adjusted to the same pH as the ambient medium, no significant change in either vacuolar or cytoplasmic pH was observed during the 35 minute experiment. These results were confirmed in experiments with pH-sensitive fluorescent dyes. We conclude that suspension-cultured soybean cells do not respond to elicitation by significantly changing the pH of their vacuolar or cytoplasmic compartments.

Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae.

Bacterial attachment is believed to be an early step in gram-negative nosocomial pneumonia. The frequency of fimbria-associated adhesins among respiratory pathogens has not been studied in detail. In this study isolates belonging to the family Enterobacteriaceae, prospectively obtained from intensive care unit patients who were suspected of having nosocomial pneumonia, were examined for fimbria-associated adhesins. Type 3, P, type 1, and other fimbrial phenotypes were identified by specific hemagglutination and electron microscopy. The Klebsiella type 3 fimbrial phenotype was further characterized by using a monoclonal antibody. Also, both type 3 and Escherichia coli P fimbrial genotypes were detected by using DNA colony blot assays. The frequencies of genera or species isolated were as follows: Enterobacter (38.6%), Klebsiella (26.8%), Serratia (17.7%), E. coli (13%), and Proteus (5.2%). Isolates of Klebsiella oxytoca, K. pneumoniae, and Enterobacter cloacae most commonly possessed the type 3 fimbrial phenotype and genotype. The phenotype and genotype for E. coli P fimbriae (46.2 and 50%, respectively), a known pathogenic determinant in the urinary tract, were detected more frequently than expected. In addition, a previously unspecified hemagglutinin that was specific for porcine erythrocytes was almost uniformly expressed among isolates of Enterobacter aerogenes. Finally, the expression of the type 1 fimbrial phenotype was widely detected among the isolates tested but notably absent among K. oxytoca and Proteus mirabilis isolates. The frequency of the various fimbrial types identified suggests a role for these bacterial organelles in adherence to respiratory epithelia.

Biotin-mediated delivery of exogenous macromolecules into soybean cells.

We have demonstrated that attachment of biotin to a variety of macromolecules allows the uptake of those macromolecules into cultured soybean cells (Glycine max Merr cv Kent). Macromolecules that were nondestructively delivered into intact cells in large numbers (>10(6)/cell) by this technique include bovine insulin (M(r) about 5,700), bovine ribonuclease (M(r) about 14,000), human hemoglobin (M(r) about 64,000), and bovine serum albumin (M(r) about 68,000). It is hypothesized that this methodology may be useful for delivering antibodies, toxins, enzymes, and genetic material into living plant cells without requiring prior removal of the cell wall or infection with Agrobacterium.

Receptor-Mediated Endocytosis in Plant Cells.

We have employed fluorescein and 125l-labeled elicitors of the defense response in soybeans to monitor the cellular distribution and movement of elicitors following their addition to a soybean cell suspension culture. Our results indicate that the macromolecular elicitors first bind to the cell surface and then internalize in a temperature- and energy-dependent endocytotic process. Within a few hours, virtually all of the elicitor is concentrated in the major vacuole or tonoplast of the cell. Nonspecific (control) proteins neither bound to the cell surface nor internalized in parallel assays.