Packing in protein cores.

Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of [Formula: see text], which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

5-hydroxytryptamine-stimulated accumulation of 1,2-diacylglycerol in the rabbit basilar artery: a role for protein kinase C in smooth muscle contraction.

1. 5-Hydroxytryptamine (5-HT) produced a concentration-dependent increase in the membrane concentration of 1,2-diacylglycerol (DG) in the rabbit isolated basilar artery, but did not stimulate the hydrolysis of membrane phosphoinositide. 2. The 5-HT-induced accumulation of DG could be blocked with the putative phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; 70 microM), but not with the protein kinase C inhibitor, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H7; 50 microM). 3. Direct stimulation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) produced sustained smooth muscle contraction which was fairly rapid in onset and could be reversed by H7 but not by NCDC. The inactive phorbol, 4 alpha phorbol 12,13-dideceonate, did not produce contraction in the basilar artery. 4. 5-HT-induced contractions (1 nM-100 microM) were blocked or greatly reduced in the presence of the protein kinase inhibitor H7 or polymyxin B, and with the phospholipase C inhibitor, NCDC. The concentrations of these inhibitors which abolished contraction to 5-HT, did not alter smooth muscle contraction produced in response to 30 mM K(+)-physiological salt solution (PSS). 5. These data suggest that DG production and the subsequent activation of PKC forms an important component of the cerebrovascular contractile response to 5-HT. As the DG does not appear to arise from membrane phosphatidylinositol, it appears that 5-HT can stimulate the production of this second messenger in cerebral arteries by a mechanism which is different from peripheral arteries.

Endothelium-dependent contractile responses to 5-hydroxytryptamine in the rabbit basilar artery.

1 5-Hydroxytryptamine (5-HT) and 5-carboxamidotryptamine (5-CT) stimulated additional, endothelium-dependent contractions in rabbit isolated basilar arteries which had been submaximally contracted with either histamine or potassium chloride. 2 The additional contractions to 5-HT were not altered by the 5-HT2 antagonist, ketanserin (1 microM), but were abolished in the presence of the cyclo-oxygenase inhibitor indomethacin (3 microM). 3 The additional smooth muscle contraction stimulated by 5-HT was increased in the presence of the competitive substrate inhibitor for nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4 Neither of the selective 5-HT agonists, 8-hydroxy-dipropylaminotetralin (8-OH DPAT) or alpha-methyl 5-HT stimulated endothelium-dependent contraction, but these agonists did reduce the rate at which histamine-induced tension spontaneously declined. This effect represented a direct action on the smooth muscle cells, as it was independent of the presence of endothelial cells. 5 Smooth muscle relaxation was not obtained in response to 5-HT, whether or not indomethacin was present to block endothelium-dependent contraction. None of the other selective 5-HT agonists, 5-CT, 8-OH DPAT or alpha-methyl 5-HT produced endothelium-dependent smooth muscle relaxation, when applied against a background of contraction. 6 These data show that endothelium-dependent smooth muscle contraction can be produced by stimulating 5-HT receptors in the partially contracted rabbit basilar artery. Similar contraction to 5-CT indicates an involvement by 5-HT1 receptors. The susceptibility of the contractions to indomethacin suggest they are mediated by a metabolite of arachidonic acid.

Comparison of different scoring systems for immunohistochemical staining.

AIM: To carry out an objective assessment of two systems of scoring immunohistochemical staining, evaluating interobserver and intraobserver error. METHODS: 92 cervical tumours underwent immunohistochemical staining for p53 and epidermal growth factor receptor. Staining was assessed using two methods: a standard 4 point scale and a descriptive method, performed by three observers. Interobserver and intraobserver error were assessed for both scoring methods. RESULTS: In terms of interobserver error between three observers, no difference was found between a simple 4 point scale method of evaluation and the use of a highly circumscribed method. In all evaluations, interobserver error was scored as moderate (kappa w 0.48-0.49). However, evaluation of immunohistochemical staining by a panel of observers led to a marked improvement in the interobserver error scores (kappa w 0.63). CONCLUSIONS: There should be standardisation of immunohistochemical staining and scoring methods. More attention should be paid to measurement of interobserver and intraobserver error in studies. Use of a panel of tissue control slides and consensus scoring by several observers can lead to improvement in reproducibility.

Reoperative thyroid surgery.

BACKGROUND: Patients with thyroid cancer are sometimes denied repeat thyroid operations for fear of an increased risk of complications. METHODS: We therefore reviewed our experience in 114 patients with benign or malignant thyroid tumors who underwent 116 thyroid reoperations with or without other procedures. All patients had undergone at least one prior thyroid operation and 16 patients had undergone from two to four thyroid operations before referral. The initial histologic diagnosis before reoperation was thyroid carcinoma in 79 patients, papillary carcinoma in 47 patients, follicular carcinoma in 17 patients, medullary carcinoma in 9 patients, and Hürthle cell carcinoma in 6 patients. Benign disease was present in 35 patients. In 62 patients with cancer, reoperations were performed because of suspected persistent or recurrent disease; one of these patients underwent two reoperations by us. In 17 patients reoperation was to complete total thyroidectomy, primarily so that radioactive iodine could be used to scan for and treat metastatic disease. RESULTS: Among the 116 reoperations, 102 were completion total thyroidectomy, 8 were near-total or subtotal thyroidectomy, and 6 were completion lobectomy. Histologic examination at reoperation revealed thyroid carcinoma in 51 cases (64%) among the 79 patients who had undergone 80 operations for previous thyroid cancer. Recurrent or persistent cancer was present in 49 of 63 (78%) reoperations for patients with papillary, medullary, and Hürthle cell cancer but in only 2 of 17 (12%) patients with follicular cancer. Cancer also occurred in 8 cases (22%) of the 36 reoperations in 35 patients who initially had benign lesions. Complications included one permanent and one transient palsy of the recurrent laryngeal nerve; both occurred on the side of a previous partial or subtotal lobectomy. Other complications included temporary hypoparathyroidism in four patients, seromas in two patients, and a keloid in one patient. CONCLUSIONS: This study documents that reoperations can be performed with minimal morbidity. Thus patients should not be denied the chance to undergo removal of a persistent tumor or the remnant normal thyroid tissue because of the fear of complications.

Ca2+ channel antagonists and inhibition of protein kinase C each block contraction but not depolarization to 5-hydroxytryptamine in the rabbit basilar artery.

The Ca2+ channel antagonists nifedipine and verapamil each significantly inhibited (50-100%) the smooth muscle contraction induced in response to either 5-hydroxytryptamine (1 microM, 5-HT) or 20 mM K+ (K(+)-physiological salt solution) in the basilar artery. Simultaneous measurements of smooth muscle membrane potential showed that changes in potential were not modified at this time. A similar inhibitory action against the smooth muscle contraction but not the depolarization to 5-HT was obtained with the putative protein kinase C and phospholipase C inhibitors, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (10 microM, H7) and 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (70 microM, NCDC). These data indicate that 5-HT-induced Ca2+ influx through voltage sensitive channels is important for smooth muscle contraction but not depolarization in the rabbit basilar artery.

Interfacial tension in phase-separated gelatin/dextran aqueous mixtures.

The effect of solute concentrations on interfacial tension was investigated in phase-separated mixtures of dextran and gelatin over a range of concentrations that covered different tie-lines and different positions on one tie-line. The investigations were carried out using equilibrated gelatin-rich and dextran-rich phases in a computer-controlled Couette device at 40 degrees C (above the gelation point of gelatin) and interfacial tensions were measured using the retracting drop method. The results show that the interfacial tension can be related to the length of the tie-line or to the difference in the concentration of dextran (or gelatin) in the separated phases. Interfacial tension increases as either of these parameters increases. For concentrations lying on any single tie-line, the interfacial tension is constant and independent of the concentration of biopolymers. Also, the addition of small amounts of low molecular weight dextran to a dextran-rich phase does not significantly affect the interfacial tension between the gelatine-rich and dextran-rich phases. Experimental results were also compared with theoretical predictions of the interfacial tension using a Flory-Huggins based analysis of the measured tie-line data. Reasonable agreement was found between predicted and measured values, indicating that this approach captures the basic physics of the system.

Shear modulus-temperature meltdown profiles of gelatin and pectin gels. A cascade theory description.

In this paper, a cascade theory approach to biopolymer gelation is developed to describe variation of the shear modulus with temperature for thermoreversible gels. The broadness of this 'melting transition' is seen to depend crucially on the enthalpy of cross-linking, while the critical gel melting temperature is determined by additional factors such as the entropy of cross-linking, the polymer concentration, the molecular weight, and the number of cross-linking sites. On using the model to fit experimental data for specific gelatin and pectin systems, it was found that the behaviour of gelatin (sharp melting transition and low melting temperature) is a consequence of the large negative enthalpy and entropy of cross-linking, while, in contrast, the broad melting transition and high melting point of the pectin system are consistent with much smaller negative values for these parameters.

Calcium-induced gelation of low methoxy pectin solutions--thermodynamic and rheological considerations.

Rheological changes occurring during the gelation of low methoxy pectins in the presence of calcium ions were investigated. The onset of gelation was found to correspond to the same macroscopic rheology (i.e. value of G') whatever the gelling conditions. Sol-gel diagrams for calcium pectin systems were mapped out in terms of calcium level, temperature and pectin concentration. The thermodynamic parameters for the cross-linking process were derived.

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH beta-lactoglobulin gels.

Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2θ=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

Current manpower pool in cytotechnology.

Data are presented on the manpower shortage in the field of cytotechnology and the lack of sufficient training programs to meet current needs, and the fiscal bases of these problems are discussed. The workload placed on cytotechnologists is also analyzed in financial terms, emphasizing the pressures for high productivity in frequently less-than-optimal conditions. Screening accuracy is also considered. All of these issues need attention to produce adequate numbers of accurate screeners.

Early endocervical glandular neoplasia. I. Histomorphology and cytomorphology.

A study is presented of 36 cases of early endocervical glandular neoplasia comprising three specific entities: endocervical adenocarcinoma in situ, microinvasive adenocarcinoma and adenocarcinoma limited to a single polypoid structure. A clinical profile is developed that reveals a typical patient population. The histomorphologic and cytomorphologic features of early endocervical glandular neoplasia are discussed in detail. The consistently reproducible cytologic appearance described may be accurately detected microscopically. Early detection of endocervical adenocarcinoma could produce a dramatic decrease in patient morbidity and mortality, such as has occurred with squamous epithelial malignancies.

Early endocervical glandular neoplasia. II. Morphometric analysis of the cells.

A morphometric analysis was performed on the cells of early endocervical glandular neoplasia, including adenocarcinoma in situ and microinvasive adenocarcinoma, in cytologic preparations. The measurements were compared with those of cells from the normal endocervix and cells from other lesions that enter into the differential diagnosis. The morphometric profile developed for the cells of early glandular neoplasia proved to be useful, reliable and reproducible. Not only could the cells of normal endocervical samples be distinguished from those of early endocervical glandular neoplasia, but the measurements also seemed capable of making the visually difficult distinction between adenocarcinoma in situ and microinvasive adenocarcinoma. The latter point requires further confirmation since only a few cases of microinvasive adenocarcinoma were available for study.

A morphometric study of primary adenocarcinoma of the vagina.

Primary adenocarcinoma of the vagina accounts for approximately 1% of all invasive carcinomas of the female genital tract. This paper reports on a study of four cases of primary adenocarcinoma (including one with in utero diethylstilbestrol exposure) of the vagina from the files of the Medical University of South Carolina. Both tissue sections and cytologic preparations from each case were evaluated in order to delineate the morphologic characteristics of the cells that compose the lesions. Histologically, the lesions primarily presented the tubular cystic pattern; solid and papillary growth patterns were also seen in one case. The constituent cells included clear cells, hobnail cells and secretory and nonsecretory tubular cystic cells. All specimens were examined for the following parameters: cell population, nuclear area, cytoplasmic area, nuclear/cytoplasmic (N/C) proportions, nuclear and cytoplasmic characteristics. While a fairly detailed profile of the morphologic criteria for hobnail cells was developed, the cellular profiles of the other malignant glandular cells originating in primary vaginal adenocarcinoma remain less certain.

Papillary endothelial hyperplasia (Masson's tumour) of the maxillary sinus.

In 1923, Masson described a neoplastic process consisting of papillary hyperplasia of the endothelial cells, with a consequent obliteration of the vascular lumen, followed later by degenerative changes. Masson coined the term vegetant intravascular haemangioendothelioma, however, these days it is more commonly known as papillary endothelial hyperplasia (PEH), or by the pseudonym, Masson's tumour. Although relatively rare, there are numerous accounts of PEH in the literature, describing its predilection for the head and neck region. Our case report describes the finding of a PEH within the paranasal sinuses, a site not previously mentioned even in the largest of series found on literature search. We will then discuss the relevant histological features of the lesion, and its natural history.

COX-1 and COX-2 expression in stage I and II invasive cervical carcinoma: relationship to disease relapse and long-term survival.

COX-1 and COX-2 are members of the cyclooxygenase (COX) family, which influence tumor invasion and apoptosis. The objective of the study was to assess the relationship between COX-1 and COX-2 expression in early-stage disease and subsequent disease relapse and long-term survival. Women with FIGO stage I and II cervical carcinoma, younger than 50 years, treated between 1981 and 1990 were included. COX-1 and COX-2 expressions in the tumors were assessed by immunohistochemistry. COX-1 and COX-2 were expressed in 61% (17/28) and 57% (16/28) of tumors, respectively. COX-1 nonexpressers showed an improved overall survival compared to expressers (log-rank test, P= 0.09). There was no significant difference in the overall survival in COX-2 nonexpressers compared to expressers (P= 0.6). Out of eight women with disease relapse, COX-1 or COX-2 expression was noted in six of eight tumors, and both were expressed in five of eight tumors. Our preliminary data suggest an adverse prognosis with COX-1 expression in early-stage cervical carcinoma and a trend toward COX-1 expression in disease relapse. The association between COX-2 expression and a worse prognosis was not proven in this study.

Funding support for population research.

PIP: Overview of the current status of population research. 4 categories of population research are defined: 1) reproductive sciences; 2) contraceptive development; 3) contraceptive evaluation; and 4) social and behavioral sciences. Following a brief description of the types of research being carried out in these areas, recent trends in funding both for research and development are reviewed, including funding by the Ford Foundation, Rockefeller Foundation, The Population Council, The Andrew W. Mellon Foundation, the US Dept. of Health and Human Services, and USAID. Government funding is seen as becoming increasingly important in maintaining research and development as private sources of funding become more scarce. Along with changes in funding patterns, there have been changes in research emphasis within private funding groups. The reproductive sciences have continued to receive the largest support, followed closely by the social and behavioral sciences. Promising new research is underway on the development of new contraceptives. Emphasis is on increased safety, efficacy and convenience, with male contraceptives being given increased attention.^ieng