Unprecedented overmetabolism of a porphyrinogen substrate by coproporphyrinogen oxidase

Harderoporphyrinogen-I is metabolized by avian hemolysate preparations of coproporphyrinogen oxidase to give a trivinylic product; this unprecedented 'overmetabolism' of the porphyrinogen substrate provides strong support for a proposed model of the active site of this poorly understood enzyme.     

Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

Background  

Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of B. burgdorferi in infected mouse tissues.     

Synthetic peptides that mimic the adhesive recognition signal of fibronectin: differential effects on cell-cell and cell-substratum adhesion in embryonic chick cells

Although fibronectin has been implicated in cell-cell as well as cell-substratum interactions, most experimentation has focused on cell-substratum interactions of fibroblasts. We have examined the effect of the specific peptide GRGDS derived from the cell-binding sequence of fibronectin upon cell-cell and cell-substratum interactions using embryonic cells and tissues. Embryonic chick segmental plate cells undergo compaction (i.e., increased cell-cell adhesion) during the early stages of somitogenesis. Fibronectin has been implicated in this increase in cell-cell interaction. In contrast, precardiac mesoderm undergoes directional migration upon a fibronectin-rich substratum, exhibiting both cell-cell and cell-substratum interactions. The segmental plate cells, which are the precursors of embryonic somites, normally show very little cell-cell or cell-substratum interaction in culture. These cells exhibit a striking increase in intercellular adhesion, but exhibit no cell-substratum adhesion, in the presence of relatively low concentrations of the fibronectin-derived peptide GRGDS. Somite cells, which normally exhibit both cell-cell and cell-substratum adhesion in culture, show complete inhibition of cell-substratum adhesion in the presence of this peptide. Precardiac mesoderm, which normally exhibits both cell-cell and cell-substratum adhesion in culture, shows a marked inhibition of both processes in the presence of GRGDS. Since the finding that a monovalent competitive inhibitor of fibronectin binding can stimulate cell-cell adhesion was unexpected, we propose a "trigger" hypothesis, whereby the peptide recognition signal acts as a specific signal or trigger for the morphogenetic process of compaction. There is a striking specificity to this effect, since synthetic peptides with even conservative changes in the amino acid sequence have no effect. Finally, we find that under certain conditions the effect of the specific peptide is lost in 6-8 hr and the cells resume cell-substratum interactions or, in the case of the segmental plate cells, revert from the compacted state and exhibit a substantial decrease in cell-cell adhesion. Our studies indicate the diversity of cell and tissue responses possible when even a single peptide inhibitor of adhesion, and we have identified the first known activating effect of a fibronectin peptide on cell behavior and differentiation.     

Identification of major autolytic cleavage sites in the regulatory subunit of vascular calpain II. A comparison of partial amino-terminal sequences to deduced sequence from complementary DNA

Purified calpain II from vascular smooth muscle is a heterodimer consisting of catalytic (Mr = 76,000) and regulatory (Mr = 30,000) subunits. In the presence of Ca2+, the regulatory subunit undergoes stepwise autolysis resulting in enzyme activation. By slowing autoproteolysis, we identified major autolytic intermediates of the regulatory subunit. Gas-phase sequencing of the regulatory subunit and its autolytic fragments revealed that the NH2-terminus of the Mr = 30,000 form was blocked, whereas each fragment yielded a unique amino acid sequence, suggesting that autolysis proceeds in an NH2- to COOH-terminal direction. By comparison of actual amino acid sequences of autolytic cleavage intermediates to the full sequence deduced from cDNA, we have identified the major autolytic cleavage sites. Three different peptide bonds were cleaved, with neutral amino acids predominating on both sides of the peptide bond hydrolyzed. Importantly, leucine or isoleucine was identified in the second position upstream from the cleavage site in all three autolytic sequences. The presence of an upstream leucine residue in the autolytic cleavage sequence is reminiscent of the structure of potent microbial and synthetic peptide inhibitors of calpain.     

Somitogenesis in the mouse embryo

This report describes the initiation of somitogenesis in the mouse embryo. Correlations are made with fibronectin distribution around the unsegmented mesoderm and the distribution of cytoskeletal elements within the cells as they undergo morphogenetic movements. The same temporal and topological changes in fibronectin, laminin, and cytoskeletal elements are seen in mouse somitogenesis as in the chick embryo. A notable exception is that the epithelial stage of somitogenesis in the mouse does not form a closed vesicle as it does in the chick. In the mouse the mesial portion of the forming somite does not become epithelial before the migration of sclerotomal cells.     

Generation of glyoxylate in methylotrophic bacteria

The mode of glyoxylate production from acetyl-CoA was investigated in three strains of methylotrophic bacteria,Pseudomonas MA,Pseudomonas AM1 and organism PAR. This investigation was prompted by the recently reported discovery of a homoisocitrate lyase in methylotrophic bacteria and the suggested involvement of this novel enzyme in assimilation of C₁ and C₂ compounds as part of a homoisocitrate-glyoxylate cycle. We were unable to detect cleavage of any of the four stereoisomers of homoisocitric acid by cell-free extracts of C₁-or C₂-grown bacteria. Extracts of C₁-grown bacteria did not catalyze condensation of glyoxylate with glutarate or production of glyoxylate from acetyl-CoA and 2-ketoglutarate. Extracts of C₁-grownPseudomonas MA catalyzed cleavage of isocitrate;threo-homoisocitrate was a potent competitive inhibitor of this reaction. These results indicate that homoisocitrate cleavage does not occur in any of the methylotrophs tested. The pathway for oxidation of acetyl-CoA to glyoxylate inPseudomonas AM1 and organism PAR therefore remains obscure.     

Breast Reconstruction for Malignant or Premalignant Disease

In ten patients breast reconstruction was done after surgical treatment for a premalignant or malignant breast disease. In six of these, prophylactic subcutaneous mastectomy and implant reconstruction were carried out, and in the remaining four reconstruction was done after simple or modified radical mastectomy. It is suggested that these procedures should be considered by those physicians and surgeons who undertake evaluation and treatment of breast disease in women. Breast reconstruction should be considered and offered to patients who suffer from the severe personal and emotional trauma attendant to surgical operation for breast disease.     

Benefits of the CO2 laser in oral hemangioma excision

Twenty-one patients with small localized oral cavity hemangiomas of the lips, tongue, and buccal mucosa are reported in this series. Although the lesions were not massive or high-flow/high-pressure vascular tumors, the simultaneous cutting and coagulation capabilities of the CO2 laser were demonstrated, rendering such excisional surgery more precisely and easily accomplished with a great deal less bleeding and a marked reduction in postoperative pain and edema. Many of the patients in this series were done as outpatients under local anesthesia. Results were acceptable and complications were minimal. Thus this laser is recommended both for the patient care and comfort aspects and the ability to render oral cavity hemangioma excisional surgery much more safely and easily done with a significant decrease in hospital utilization and overall medical expenses.