Structural determinants of the bifunctional corn Hageman factor inhibitor: x-ray crystal structure at 1.95 A resolution

Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13.6 kDa protein isolated from corn seeds. It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulation as well as alpha-amylases from several insect species. Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases. We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution. Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution. The two proteins are essentially identical. The proteinase binding loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the family of 25 cereal trypsin/alpha-amylase inhibitors, we have made three-dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase inhibitor 0.19 [Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T. (1997) Biochemistry 36, 13503-13511]. From an analysis of the models and a structure-based sequence analysis, we propose a testable hypothesis for the regions of these proteins which bind alpha-amylase. In the course of the investigations, we have found that the cereal trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants. This is a new addition to the group which now consists of the trypsin/alpha-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family. Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.     

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.     

Total solid phase synthesis of gamma-subunit of cGMP phosphodiesterase from bovine retina and physicochemical properties of synthetic protein

The 87-membered polypeptide with the sequence of the gamma subunit of cGMP phosphodiesterase from bovine retina rods (PDE gamma) was synthesized by the solid phase method. Two synthetic approaches, which were based on the Boc/Bzl-strategy, were used; both syntheses were carried out in a continuous-flow reactor with swellographic monitoring. In the first approach, five Arg residues were coupled in the form of Boc-Arg(Z)2-OH and the final cleavage of the peptide from the support was effected by the mixture of CF3SO2SiMe3 and thionisole in trifluoroacetic acid. There resulted a heterogeneous, ornitine-rich, and absolutely inactive peptide material which was insoluble in aqueous alkali. In the second approach, Arg(Tos) and the HF low-high cleavage procedure were used, which resulted in a homogeneous polypeptide (according to HPLC and capillary electrophoresis) that manifested correct molecular mass under ion-spray mass spectrometry and the full functional activity characteristic of the native protein. The effect of zinc salts on the PDE gamma fluorescence in solutions and on its solubility was established. This demonstrated a significant PDE gamma affinity with Zn2+ ions and appeared to be connected with the functioning of the protein in the retina cells. For the first time, the dynamics of the peptidylpolymer swelling in different solvents was studied during the synthesis of peptides with very long sequences.     

Variability analysis of HIV-1 gp120 V3 region: III. Distinctions between various sets of peptide fragments derived from the sequences belonging to different HIV-1 taxons

Distinction criterion for various sets of fixed length peptide fragments and integral distinction measure for various sets of peptide fragments with different length and start position ranges have been introduced on the base of an enumeration procedure and a point estimators for the amino acid distribution characteristics introduced previously (M. Yu. Shchelkanov, A. N. Yudin, A. V. Antonov, N. S. Starikov, A. A. Vedenov, E. V. Karamov, J. Biomol. Struct. Dyn. 15, 231-241 (1997)). Differences between 6-10-mer peptides derived from the majority of HIV-1 taxon pairs are demonstrated to be located generally in the vicinity of the V3-loop top. This validates the suitability of V3 top mimicking synthetic peptides for HIV-1 serotyping. A significant difference between E subtype V3 C-terminus peptides and the corresponding peptides derived from the other subtypes has been demonstrated. Taking into account the Langerhans' cells tropism of E subtype virus variants we have hypothesized the influence of mutations in the V3 C-terminus on HIV-1 cell tropism.     

Mitral valve anomalies obstructing left ventricular outflow

This paper reports on two cases of more uncommon types of subaortic stenosis. A 2-year-old boy was found with accessory mitral valve leaflet (AMVL) attaching to the anterior leaflet, ballooning into the subaortic ventricular septum associated with a discrete subaortic membrane. The obstruction was successfully relieved by removal of the AMVL and resection of the membrane. A 19-day-old newborn with accessory tissue on the mitral valve (AMVT) causing subaortic stenosis, subaortic ventricular septal defect (VSD) and patent ductus arteriosus was operated on successfully. Accessory tissue excision through the VSD, VSD patch closure and ductus ligation were performed.     

neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group

PURPOSE: It remains a challenge to predict which women with axillary node-negative (ANN) breast cancer at greatest risk of relapse may benefit most from adjuvant therapy. Increases in neu/erbB-2 have been implicated in breast cancer prognosis. Although overexpression has been investigated extensively, this study represents the first prospective assessment of the prognostic value of neu/erbB-2 DNA amplification in a cohort of women with newly diagnosed ANN. METHODS: A consecutive series of women was monitored for recurrence (median follow-up duration, 36 months) and tumors from 580 individuals were analyzed for amplification. The association of amplification with risk of recurrence was examined in survival analyses with traditional and histologic markers as prognostic factors. RESULTS: Neu/erbB-2 was amplified in 20% of cases. We found an increased risk of disease recurrence when neu/erbB-2 was amplified > or = twofold that persisted with adjustment for other prognostic factors (relative risk, 2.36; P = .002). We found some evidence that amplification was more important in patients who received chemotherapy compared with untreated patients. CONCLUSION: neu/erbB-2 amplification is an independent prognostic factor for risk of recurrence in ANN breast cancer. Women with tumors without neu/erbB-2 amplification have a good prognosis; aggressive therapy in this group is therefore difficult to justify. On the other hand, even with adjuvant chemotherapeutic treatment, women whose tumors exhibit neu/erbB-2 amplification have an increased risk of recurrence. We encourage a randomized trial to compare more aggressive adjuvant chemotherapy versus standard chemotherapy for ANN women whose tumors exhibit neu/erbB-2 amplification.     

The value of colonic exfoliative cytology in the diagnosis of carcinoma of the large intestine

We have attempted to summarize the current controversies regarding risk factors and preventive measures for control of arteriosclerosis and coronary heart disease. Recognizing that the genesis and development of the disease process are extremely complex and the basic knowledge is limited, it is not likely that conclusive answers to questions will be forthcoming soon which will provide more effective preventive or therapeutic measures. It might be desirable to institute educational and control program aimed at curtailing, at a young age, known AS risk factors such as heavy smoking, particularly if the family history indicates severe risk. Few will question the normal approaches to the treatment of complications of coronary heart disease by control of hypertension, elevated cholesterol, and smoking. However, great caution must be exercised when trying to institute large scale modifications in prevailing life patterns, particularly when based on indefinite risk factor studies and in the face of potentially profound and frequently unknown consequences. The unknowns of atherosclerotic heart disease risk factors, coupled with uncertainties and even doubts about protracted and expensive population studies, lead us to propose an emphasis on alternate selective approaches. We strongly believe that fundamental to progress in the field of arteriosclerosis is an amplification of preventive research efforts with stronger attention focused upon influencing the atherosclerotic processes within the arterial wall. But, more immediately, we urge systematic gathering and careful evaluation of patient data in particular population subsets which exhibit and accelerated mode of arteriosclerosis. Comparative studies of patients, particularly twins, families, and ethnic populations with redilection to early or accelerated arteriosclerosis may be extremely rewarding. Our repeated review of the enormous literature suggests that worldwide collaboration is needed to perfect more meaningful protocols as well as to correlate and critically evaluate existing data provided by population studies of this insidious disease process which represents an evermounting burden to society.     

Colonic anastomosis-a new approach: preliminary report

A case of fetal death from abruptio placentae is reported in which the placenta was the seat of multiple chorangiomata. This association is believed not to have been fortuitous and a possible mechanism is suggested by which chorangioma may contribute to the pathogenesis of placental abruption. The incidence, morphological variation and clinical manifestations of placental chorangiomata are briefly discussed.