Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

Construction of an ordered overlapping library of bacteriophage P1 DNA in phage vector lambda D69

A library of bacteriophage P1 DNA was constructed in the phage vector lambda D69. The DNA of some 150 randomly chosen lambda-P1 hybrid phages containing P1 DNA fragments 5-10 kb in size was analyzed by restriction endonuclease digestion using enzymes EcoRI, BglII, and BamHI that cleave P1 DNA at known positions on the physical map of P1. Approximately one third of the phages contained P1 DNA inserts having two or more restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with respect to the physical map. Genetic tests allowed detection of lambda-P1 hybrid phages possessing inserts with functional P1 ban and CmR genes. A subset of 18 phages was analyzed in more detail; their P1 DNA inserts comprise an ordered collection of overlapping P1 DNA fragments that cover almost 98% of the P1 genome.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Molecular diagnosis of Duchenne and Becker muscular dystrophies. Current data

Carrier diagnosis and prenatal diagnosis of Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD) has become possible using some twenty RFLPs detected by more than a dozen Xp21 probes that are either intragenic or flanking the disease locus. Results from familial studies on 88 DMD and BM families stress important considerations concerning a priori and final risks, individuals necessary for the identification of the phase, and the different strategies that can be applied, regardless of whether the study concerns an on-going pregnancy or a carrier-status determination, and whether the patient is at high or low risk. Finally, multiple sources of difficulties in interpreting the results depend on a) the occurrence of new mutations that must be traced; b) the existence of meiotic recombination; c) the necessity, in some instances, of relying upon the sole identification of the paternal X. These considerations emphasize the characteristics and the important limitations of this type of methodology.     

Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.     

Fourier transform infrared investigation of the Escherichia coli methionine aporepressor

This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli. Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E. coli Met aporepressor. The secondary structure of the native bacterial protein was derived by analysis of the amide I mode. The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures. The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%). The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%). The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein.     

Spatial organization of CaATPase molecules in sarcoplasmic reticulum vesicles

Fluorescence intensity, polarization, and (Ca2+-Mg2+)-ATPase (CaATPase) activity were measured for sarcoplasmic reticulum (SR) CaATPase with varying amounts of fluorescein isothiocyanate (FITC) attached at a specific site at or near the ATP binding site. The stoichiometry of attached FITC was proportional to the inhibition of ATPase activity, consistent with the independent labeling of one FITC site per CaATPase molecule. Polarization measurements on vesicular CaATPase indicated the occurrence of energy-transfer depolarization that increased as the fraction of binding sites labeled by FITC increased. Addition of the nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) eliminated the energy-transfer depolarization for all degrees of labeling with little direct effect on the attached FITC molecule. Fluorescence polarization measurements on sizing-column-purified FITC-labeled CaATPase in the presence of 30 mM C12E9 indicated that the sample consisted of homogeneous monomeric CaATPase. The attached FITC molecule was not sensitive to the bulk viscosity for either the vesicular or the detergent-solubilized CaATPase. The midpoints of the transition from vesicular to monomeric CaATPase as a function of increasing detergent concentration were determined from fluorescence polarization and light-scattering measurements. The dependence of these midpoints on the CaATPase concentration indicated a stoichiometry of 262 +/- 35 molecules of C12E9 per CaATPase in the detergent-protein complex. Both measurements gave the same result. The decrease of fluorescence polarization with increasing saturation of the FITC binding sites for vesicular and detergent-solubilized CaATPase was analyzed in terms of energy-transfer depolarization to determine the spatial arrangements of CaATPase molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure analysis proves that protein phosphatases 2C1 and 2C2 are isozymes

The two recently discovered forms of protein phosphatase 2C, termed 2C1 and 2C2, were digested with CNBr or trypsin, and several peptides corresponding to two regions of the protein were sequenced. These studies revealed close homology between the two enzymes with 49 identities over the 62 residues that could be compared directly. The results establish that protein phosphatases 2C1 and 2C2 are the products of different genes. The C-terminus of protein phosphatase 2C2 has also been identified.     

Reconstructive surgery for immunosuppressed organ-transplant recipients

Prolonged vascularized organ allograft survival and an improved quality of life are now possible for many transplant recipients. These advances are due largely to greater understanding of the immune response, the development of potent immunosuppressive agents (cyclosporin A), and improved surgical techniques. Thus more of these patients may require surgical procedures related or unrelated to their original operation, and the plastic surgeon, among other specialists, should be aware of the special problems of the immunocompromised transplant recipient who needs to undergo reconstructive surgery. We report our experience with 15 kidney, heart, and liver transplant recipients who required reconstructive surgery for a variety of conditions. The combined team approach by reconstructive and transplant surgeons is described, as well as the perioperative drug protocol and the special problems that immunosuppressed transplant recipients present. We conclude that these patients can successfully undergo major reconstructive procedures as long as the plastic surgeon not only performs technically flawless surgery, but also familiarizes himself or herself with the special problems of the immunosuppressed host, including the ever-present risk of sepsis and delayed and impaired wound healing, the potential for acute Addisonian crisis, and the possibility of multiple complicating comorbid conditions.     

Analysis of fibrogenic processes in denervated tissues of spinal cord injury patients

To test the hypothesis that altered collagen metabolism is a contributing factor in the apparent delayed wound healing in denervated regions of spinal cord injury (SCI) patients, a tissue implant (PVA) was used to directly measure collagen deposition. Sterile PVA implants were placed subcutaneously in the inner aspect of the upper arm above the cord injury (innervated) and in the inner aspect of the upper leg below the cord injury (denervated) of 20 spinal cord injury patients and compared to eight healthy volunteers. On day 14, the implants were removed and analyzed histologically by trichrome stain and biochemically for hydroxyproline as a measure of collagen deposition. No remarkable histologic differences were observed in the sponge material removed from the upper regions compared to the lower denervated regions of the spinal cord injury patients. Sponges from both areas were infiltrated with fibroblasts containing well-developed rough endoplasmic reticulum and large quantities of trichrome-positive collagen. Likewise, upper and lower histology of controls was identical and nondistinguishable from the corresponding sections obtained from the spinal cord injury patients. Quantitation of the hydroxyproline in the arms of the spinal cord injury patients (n = 20) showed 4.3 +/- 0.7 nmol hydroxyproline per milligram of sponge compared to 4.1 +/- 0.4 nmol/mg in the denervated regions of the lower limb. The hydroxyproline content in the arms of control volunteers was 5.2 +/- 0.7 nmol/mg compared to 3.9 +/- 0.8 nmol/mg in the leg (n = 8). These observations suggest that fibrogenic processes in denervated regions are not reduced significantly compared to innervated regions.