Conservation of PDX-1 structure, function, and expression in zebrafish.

Development of the mammalian pancreas has been studied extensively in mice. The stages from budding of the pancreatic anlaga through endocrine and exocrine cell differentiation and islet formation have been described in detail. Recently, the homeodomain transcription factor PDX-1 has been identified as an important factor in the proliferation and differentiation of the pancreatic buds to form a mature pancreas. To evaluate the possibility of using zebrafish as a model for the genetic analysis of pancreas development, we have cloned and characterized PDX-1 from this organism. The deduced sequence of zebrafish PDX-1 contains 246 amino acids and is 95% identical to mammalian PDX-1 in the homeodomain. We also cloned zebrafish preproinsulin complementary DNA as a marker for islet tissue. By in situ hybridization we demonstrate that PDX-1 and insulin are coexpressed during embryonic development and in adults, although PDX-1 expression appears to be biphasic. Insulin expression apparently begins before 44 hpf, the earliest stage examined in this study. Additionally, very high levels of PDX-1 expression were observed in the pyloric caeca, the accessory digestive organs that also are derived from the proximal region of the intestine in teleosts. Finally, our data show that the evolutionary conservation of zebrafish PDX-1 extends to its DNA binding properties. Zebrafish PDX-1 was equally as effective as mouse PDX-1 in stimulating insulin gene transcription, and maximum promoter activation was dependent on the presence of four intact A elements. The demonstration of this capability suggests that transcriptional regulatory mechanisms that control pancreatic development and insulin gene expression have been conserved among vertebrates.

Processing of wild-type and mutant proinsulin-like growth factor-IA by subtilisin-related proprotein convertases.

Insulin-like growth factor I (IGF-I) is required for normal embryonic development and postnatal growth. Like most hormones and growth factors, IGF-I is synthesized as a proprotein that is converted to the mature form by endoproteolysis. Processing of pro-IGF-I to mature IGF-I occurs by cleavage within the unique pentabasic processing motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg77. We have previously shown that human embryonic kidney 293 cells process pro-IGF-IA at Arg71 to generate IGF-I-(1-70) and at Arg77 to produce IGF-I-(1-76). Cleavage at each of these sites requires upstream basic residues, indicating that subtilisin-related proprotein convertases (SPCs) may be involved. In order to investigate the identity of the endogenous enzymes involved in maturation of pro-IGF-IA, we have expressed wild-type and mutant pro-IGF-IA in 293 cells and in the furin-deficient Chinese hamster ovary cell line, RPE.40. We have also co-expressed these constructs with SPCs that are thought to play a role in processing precursor proteins in the constitutive pathway: furin, PACE4, PC6A, PC6B, and LPC. The results show that furin is most active at cleaving wild-type and mutant pro-IGF-IA and can cleave these precursors at multiple sites within the pentabasic motif. PC6A and LPC are less active than furin but cleave only at Arg71. PACE4 and PC6B have very little activity on pro-IGF-IA precursors. Wild-type pro-IGF-IA was correctly processed to mature IGF-I in 10 of 10 cell lines that were tested. Since furin, PC6A, and LPC are known to have a broad pattern of tissue distribution and we have demonstrated expression of LPC in RPE.40 cells, our results suggest that these SPCs may be responsible for the endogenous pro-IGF-IA processing activity observed in a wide variety of cell lines.

Overproduction of a 37-kilodalton cytoplasmic protein homologous to NAD+-linked D-lactate dehydrogenase associated with vancomycin resistance in Staphylococcus aureus.

We previously reported the isolation of a laboratory-derived Staphylococcus aureus mutant, 523k, that has constitutive low-level resistance to vancomycin (MIC = 5 micrograms/ml) and teicoplanin (MIC = 5 micrograms/ml) and elaborates a ca. 39-kDa cytoplasmic protein that was not detected in the parent strain 523 (MIC = 1 micrograms/ml). We have now detected the protein in strain 523 by immunoblotting with antiserum raised against the protein. Consistent with our initial observations, densitometric analysis of the immunoblots revealed an increased production of the protein in 523k compared with that of the susceptible parent 523. The 5' region of the gene encoding the protein of interest was identified by nucleotide sequencing a PCR product amplified from the genome of 523k with degenerate primers designed to encode the amino acid sequence of proteolytic peptides obtained from the protein. The remainder of the gene was identified by library screening, PCR, and nucleotide sequencing. The gene encodes a 36.7-kDa protein with homology to a family of bacterial NAD+-dependent, D-specific 2-hydroxyacid dehydrogenases which includes both D-lactate dehydrogenase and the enterococcal vancomycin resistance protein VanH and is therefore designated ddh. Increased production of the product of ddh, Ddh, was associated with increased D-lactate dehydrogenase activity in 523k, a finding which suggested that Ddh is likely to be the D-lactate dehydrogenase previously identified in S. aureus. The increased D-lactate dehydrogenase activity in strain 523k and the structural similarities among Ddh, D-lactate dehydrogenase, and VanH suggest that overproduction of Ddh might play a role in vancomycin resistance in this strain.

Characterization of Staphylococcus aureus isolates with decreased susceptibility to vancomycin and teicoplanin: isolation and purification of a constitutively produced protein associated with decreased susceptibility.

"Derivative isolates" with 4- to 8-fold and 8- to 16-fold increases in MICs of vancomycin and teicoplanin, respectively, were selected from 2 susceptible clinical isolates of Staphylococcus aureus by serial incubation in low-level vancomycin. A protein of approximately 39 kDa was demonstrable in the cytoplasmic fraction and occasionally in the membrane fraction by SDS-PAGE of both derivatives. This protein was purified by DEAE chromatography, preparative SDS-PAGE, and electroelution. Derivative bacteria were larger on transmission electron microscopy, had thicker cell walls, and had changes in colony morphology on solid media. Further evidence for cell wall reorganization included loss of phage and capsular typing, decreased susceptibility to lysostaphin/lysozyme killing, and changes in condition for detection of optimal coagulase activity. The mechanism of decreased susceptibility to glycopeptide antibiotics among S. aureus derivative isolates is uncertain. The production of the approximately 39-kDa cytoplasmic protein and cell wall reorganization may mediate changed affinity of glycopeptide-peptidoglycan binding or impairment of glycopeptide access to its cell wall target.