Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate.

Phloem exudate from squash fruit contains heat-inactivated material which inhibits pepsin activity. This inhibitory activity was purified by mild acid treatment, chromatography on trypsin-agarose, Sephadex G-75 and reverse-phase HPLC, resulting in the elution of three peaks with pepsin-inhibitory activity. N-terminal sequencing indicated a common sequence of MGPGPAIGEVIG and the presence of minor species with seven- or two-amino-acid N-terminal extensions beyond this point. Microheterogeneity in this end sequence was exhibited within and between two preparations. Internal sequencing of a major peak after a trypsin digestion gave the sequence FYNVVVLEK. The common N-terminal sequence was used to design a degenerate primer for 3' rapid amplification of cDNA ends and cDNA clones encoding two isoforms of the inhibitor were obtained. The open reading frames of both cDNAs encoded proteins (96% identical) which contained the experimentally determined internal sequence. The amino acid content calculated from the predicted amino acid sequence was very similar to that measured by amino acid analysis of the purified inhibitor. The two predicted amino acid sequences (96 residues) had neither similarity to any other aspartic proteinase inhibitor nor similarity to any other protein. The inhibitors have a molecular mass of 10,552 Da, measured by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and approximately 10,000 Da by SDS/PAGE, and behave as dimers of approximately 21,000 Da during chromatography on Superdex G-75 gel-filtration medium. The calculated molecular masses from the predicted amino acid sequences were 10,551 Da and 10,527 Da. The inhibitor was capable of inhibiting pepsin (Ki = 2 nM) and a secreted aspartic proteinase from the fungus Glomerella cingulata (Ki = 20 nM). The inhibitor, which is stable over acid and neutral pH, has been named squash aspartic proteinase inhibitor (SQAPI).

Regulation of the secretion of Rhizopus oligosporus extracellular carboxyl proteinase.

Secretion of the extracellular Rhizopus carboxyl proteinase (EC 3.4.23.6) by Rhizopus oligosporus is repressed in the presence of low-molecular-mass sources of nitrogen, sulphur and carbon. Proteinase is secreted when the medium is deficient in any one of these three nutrients. In the case of nitrogen metabolite repression, control is at the level of transcription. Induction of proteinase secretion by exogenous protein does not occur in any of the media examined.

Effects of ischemia and hemoglobin on vascular function in isolated rabbit aortas.

The vascular endothelium is important in the modulation of vascular tone via production of endothelium-derived relaxing and contracting factors. The abdominal aortas of five groups of rabbits were subjected to varying lengths of ischemia (0, 1, 2, 3, or 4 hours), removed, sectioned into transverse rings, and placed in tissue baths containing Krebs' buffer at 37 degrees C and aerated with 95% O2/5% CO2. After equilibration the rings were tested for endothelium-dependent vasodilation with methacholine and nonendothelium-dependent vasodilation with nitroprusside. Endothelium-dependent relaxation as elicited by methacholine was impaired at 3 and 4 hours of ischemia but was not significantly different at 1 and 2 hours as compared to control, whereas endothelium-independent vasodilation remained normal throughout the different periods of ischemia. The addition of 1 x 10(-6) mol/L rabbit hemoglobin reduced the time needed to demonstrate significant impairment of endothelial function to 2 hours. Endothelium-independent vasodilation was not affected by hemoglobin. We conclude that hemoglobin exacerbates ischemia vascular dysfunction in the rabbit aorta.

Experimental inhalation injury with concomitant surface burn: dextran resuscitation improves lung water and oxygenation.

UNLABELLED: The role of extravascular lung water (EVLW) in the pathogenesis of inhalation injury (INH) when associated with concomitant major burn (B) remains controversial. Previous experimental models have investigated isolated INH without surface burn. This study measured the effects of isolated and combined INH on EVLW and pO2 in a porcine experimental model. The beneficial effects of early resuscitation with dextran-40 (DEX) were assessed, using a control group receiving standard Parkland formula (LR). In the first part of the study (INH vs. INH + B), a group of animals with a standardized INH was compared to a group also receiving a standardized 40% BSA third-degree surface burn (n = 8, each group). With serial measurements for 5 hours, EVLW was only modestly increased unless INH was accompanied by surface burn: 20.3 +/- 4.2 vs. 32.0 +/- 4.1 ml/kg at 5 hours (p less than 0.01). Similarly, pO2 fell much more dramatically in the INH + B group, 61 +/- 5 vs. 37 +/- 5 torr (p less than 0.05). The second part of the study compared standard Parkland crystalloid resuscitation with dextran-40 resuscitation in animals receiving a combined INH + B injury (LR vs. DEX, n = 8, each group). DEX resuscitation resulted in substantially lower accumulation of EVLW out to 5 hours, 34.1 +/- 5.0 vs. 13.1 +/- 3.0 ml/kg (p less than 0.01), and significantly better pO2, 35 +/- 5 vs. 64 +/- 4 torr (p less than 0.01). CONCLUSIONS: Inhalation injury did not dramatically increase EVLW in this animal model unless accompanied by concomitant major surface burn. The deterioration in EVLW and pO2 seen in the combined injury was significantly improved with DEX resuscitation when compared to standard crystalloid resuscitation. Further study is indicated and clinical trials may be warranted.

Iron deficiency anemia. How to diagnose and correct.

The prevalence of iron deficiency anemia has decreased in recent years because of improved dietary habits. Yet, iron deficiency anemia is still the most common anemia. Among mature adults, anemia of chronic disease is probably more common. Mean corpuscular volume and red cell distribution width, along with a peripheral smear examination, can often distinguish iron deficiency anemia from other common microcytic anemias, such as thalassemia minor. A normal serum iron level excludes iron deficiency anemia and indicates other causes for microcytic anemia. Often, a low serum iron level and total iron-binding capacity are due to chronic disease, and measurement of serum ferritin or a bone marrow stain for hemosiderin will be necessary to diagnose iron deficiency. Iron therapy to restore the red cell mass should be continued until iron stores are replenished.

Morphological and functional techniques for assessing endothelial integrity: the use of Evans blue dye, silver stains, and endothelial derived relaxing factor.

The endothelium is an important aspect of vascular function and pathology. Simple and reliable methods for assessing the presence or absence of endothelium in vascular specimens are presented. Intravenous Evans blue dye that stains endothelial denuded vessel walls in recommended for the macroscopic visualization of endothelial injury in gross intact arterial specimens. Scanning electron microscopy (SEM) is often used for investigating microscopic endothelial injury, but the differentiation of endothelial cells from underlying vessel wall structures can be difficult. By demarcating endothelial cell borders, silver stains allow for better endothelial identification by SEM. A third technique describes assessing endothelial integrity by how well it functions and involves the selective production of endothelium derived relaxing factor. As cardiovascular surgeons become more involved in basic vascular research, a review of these techniques should be appropriate and helpful.

Assessing clamp-related vascular injuries by measurement of associated vascular dysfunction.

The development of vascular clamps requires a reliable method to quantitate clamp-related vascular injuries. The degree of vessel damage usually is estimated subjectively from photomicrographs made with scanning electron microscopy. In order to test whether the use of vascular rings to assess residual vascular function may be a better method, rabbit thoracic aortas were occluded by five types of clamps: a Fogarty softjaw bulldog, a Fogarty Hydragrip, a 6-inch Satinsky clamp, an Edslab bulldog, and a silicone vessel band. Each area of clamp injury was sectioned into a vascular ring and suspended in a tissue bath. Residual vascular function was determined by contraction in response to phenylephrine and by relaxation in response to methacholine chloride and sodium nitroprusside. Morphologic studies with use of Evans blue dye and scanning electron microscopy complemented the vascular-function studies. The Fogarty bulldog clamp was the best at preserving vascular contraction and relaxation; the more crushing Satinsky clamp was the worst. The testing of vascular rings for residual function appears to be a useful technique for objectively quantitating vascular clamp-related vascular damage.

Endothelial injury and vascular dysfunction associated with the Fogarty balloon catheter.

Endothelial-derived relaxing factor is an important modulator of vascular relaxation, and its production is very sensitive to endothelial injury. The abdominal aortas of five groups of rabbits were subjected to Fogarty balloon catheterization via the femoral artery. Some rabbits were killed immediately (time 0), and others were killed at weekly intervals. At time 0, isolated vascular rings sectioned from the abdominal aortas and suspended in tissue baths failed to exhibit any endothelial-derived relaxing factor dependent vascular relaxation. Vascular relaxation progressively returned but was not normal until 4 weeks after the initial injury. Morphologic changes studied by means of Evans blue dye and by scanning electron microscopy with silver staining corresponded well with these functional changes. At time 0, the delicate endothelium was completely denuded, but it progressively regenerated over the subsequent 4 weeks. This severe injury to the vascular endothelium and the resultant vascular dysfunction may result in an increased risk of early postoperative spasm and thrombosis after the use of a Fogarty balloon catheter.

Colorectal cancer. Are adjuvant therapies beneficial?

The American Cancer Society's recommendations for screening the population at normal risk for colorectal cancer have been promoted since 1983. Additional study of the effectiveness of these recommendations is in progress, but the medical community has generally endorsed them and urges their implementation in office practice. To date, chemotherapy is not clearly indicated as adjuvant therapy, but enrollment of patients in cooperative group protocols is recommended. Use of second-look surgery to resect selected recurrent tumors has increased in recent years, in part because of the contribution of the computed tomographic scan and carcinoembryonic antigen assay to follow-up of postoperative patients. For advanced disease, technologic advances have improved the technique of hepatic artery perfusion with chemotherapeutic agents, but evidence of a survival advantage afforded by this method is still lacking. Single-agent chemotherapy with fluorouracil remains the best choice for palliating advanced disease, but hepatic artery perfusion with floxuridine also may relieve liver-dominant disease.

The cellular location of proteases in Candida albicans.

Vacuoles prepared from yeast cells of Candida albicans were enriched in proteinase ycaB (EC 3.4.21.48) but not in aminopeptidase or beta-glucosidase. Proteinase ycaB, assayed in situ, increased 1.5-fold during starvation whereas aminopeptidase activity decreased by 25%. Proteinase ycaB increased a further 1.5-fold during germ-tube formation.

Multiple lymphomatous polyposis of the gastrointestinal tract with immunologic marker studies.

We report a rare case of multiple lymphomatous polyposis of the gastrointestinal tract, with emphasis on the radiological, colonoscopic, and morphological findings. The underlying condition proved to be a poorly differentiated lymphocytic lymphoma. This is the first case of multiple lymphomatous polyposis in which immunologic marker studies are described and B cell origin confirmed. The different types of lymphoid polyps of the colon and the role of immunohistology are also discussed.

Severe hypernatremia in a patient with psychiatric illness.

A 17-year-old schizophrenic developed severe hypernatremia during a period of psychosis. The thirst-deficient abnormality that caused this hypernatremia resolved when his psychosis improved. The primary disorders causing a thirst deficiency leading to hypernatremia fall into three categories: lesions of the central nervous system, mineralocorticoid excess, and drug side effects. None of these disorders was found in our patient. We conclude that psychosis can severely impair the thirst mechanism directly.

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.

Implementing clinical pharmacy services in an outpatient oncology clinic.

The implementation of clinical pharmacy services in an outpatient oncology clinic is described. An oncologist hired by the department of internal medicine and the director of the department of pharmacy designed the program. Both departments agreed that the pharmacists should have central responsibility for the antineoplastic agents. Pharmacists for the program were selected from the existing staff. They were trained in an established i.v. therapy certification course offered by the hospital. The biweekly clinics are staffed by a team consisting of two pharmacists, three nurses, two faculty physicians, and a rotating resident physician. The pharmacists provide clinical and distributive services including patient monitoring and medication storage, delivery, preparation, administration, and disposal. A survey showed that the pharmacists were well accepted by the other members of the team. After a trial period, principles of break-even analysis and differential accounting were used to justify the costs of the program. This program will remain an integral part of this hospital's pharmaceutical services.

Effect of topical bile acids on gastric surface epithelial cells.

UNLABELLED: Attempts to assess the integrity of the gastric surface epithelial cell layer have been hampered by the lack of a quantitative index of surface cell injury. Two approaches: scanning electron microscopy and measurement of DNA efflux (DNAE) were used to assess surface cell injury produced by topical bile acids. Taurocholic acid (TC) at 0, 2.5, 5, and 10 mM concentrations in both neutral (NTS, pH = 7.0) and acid test solution (ATS, pH = 1.2) was applied to chambered vascularized wedges of proximal canine gastric mucosa for sequential 30-minute study periods. In part I surface cell morphology was assessed by scanning electron microscopy in 28 mucosae exposed to NTS, NTS + TC, ATS, or ATS + TC, and the effect of the mucolytic agent N-acetyl-L-cysteine (acetylcysteine) at pH 7.0 was studied in 10 additional mucosae. In part II DNAE, net cation fluxes, and mean potential difference (PD) per 30-minute period were measured. In 16 animals 10% acetylcysteine was applied topically after period III to dissolve gastric mucus and allow recovery of exfoliated cells. Nineteen additional mucosae were treated with acetylcysteine between each study period. RESULTS: By scanning electron microscopy, mucosae exposed to NTS, NTS + TC, or ATS maintained an intact layer of surface epithelial cells. ATS + TC induced dose-dependent exfoliation of sheets of surface cells. Compared with NTS, neither NTS + TC nor ATS altered DNAE; DNAE increased only in mucosae exposed to ATS + TC. Compared with ATS, ATS + TC increased cation fluxes and decreased PD (p less than 0.05). DNAE correlated with delta H+, delta Na+, delta K+, and PD (r = 0.95, 0.93, 0.86, and 0.89) (all p less than 0.05). Pretreatment with acetylcysteine increased Na+ flux in mucosae exposed to NTS and ATS but otherwise did not change delta H+, delta K+, DNAE, or PD. In mucosae exposed to ATS + TC, DNAE paralleled morphologic changes in the surface epithelial layer and physiologic alterations in cation fluxes and PD. Both scanning electron microscopy and measurement of DNAE appear to be useful tools in assessing surface epithelial cell injury.

T gamma (T gamma) cells suppress growth of erythroid colony-forming units in vitro in the pure red cell aplasia of B-cell chronic lymphocytic leukemia.

In vitro studies were performed in two patients with B-cell chronic lymphocytic leukemia who developed pure red cell aplasia (CLL-PRCA). During the active phase of their red cell aplasia, there was a marked reduction in the numbers of erythroid colony-forming units (CFU-E). Unfractionated sera or separated IgG fractions from these patients did not impair CFU-E proliferation from either autologous or allogeneic marrows. Increased numbers of T lymphocytes were present in marrow aspirates of these patients. Analysis of these T cells indicated that 90 and 35%, respectively, bore Fc receptors for IgG (T gamma cells). Removal of T cells by E-rosetting techniques augmented CFU-E growth in CLL-PRCA 10-fold. Similar treatment of normal marrows did not cause similar enhanced growth of CFU-E. Co-cultures of marrow T cells or T gamma cells obtained during the active phase of CLL-PRCA suppressed CFU-E growth from autologous or allogeneic marrows. After achieving drug-induced remission of the PRCA, marrow T cells were no longer inhibitory. In contrast, BFU-E (erythroid burst-forming units) or granulocyte proliferation in diffusion chambers were not suppressed by CLL-PRCA T cells. These findings suggest that the development of PRCA in B-cell CLL may result from suppression of CFU-E proliferation by T gamma cells.