Salt bridge impact on global rigidity and thermostability in thermophilic citrate synthase.

It has been suggested that structural rigidity is connected to thermostability, e.g. in enzymes from thermophilic microorganisms. We examine the importance of correctly handling salt bridges, and interactions which we term 'strong polars', when constructing the constraint network for global rigidity analysis in these systems. Through a comparison of rigidity in citrate synthases, we clarify the relationship between rigidity and thermostability. In particular, with our corrected handling of strong polar interactions, the difference in rigidity between mesophilic and thermophilic structures is detected more clearly than in previous studies. The increase in rigidity did not detract from the functional flexibility of the active site in all systems once their respective temperature range had been reached. We then examine the distribution of salt bridges in thermophiles that were previously unaccounted for in flexibility studies. We show that in hyperthermophiles these have stabilising roles in the active site; occuring in close proximity to key residues involved in catalysis and binding of the protein.

X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein.

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Inhibition of HIV-1 protease: the rigidity perspective.

MOTIVATION: HIV-1 protease is a key drug target due to its role in the life cycle of the HIV-1 virus. Rigidity analysis using the software First is a computationally inexpensive method for inferring functional information from protein crystal structures. We evaluate the rigidity of 206 high-resolution (2 Å or better) X-ray crystal structures of HIV-1 protease and compare the effects of different inhibitors binding to the enzyme. RESULTS: Inhibitor binding has little effect on the overall rigidity of the protein homodimer, including the rigidity of the active site. The principal effect of inhibitor binding on rigidity is to constrain the flexibility of the ß-hairpin flaps, which move to allow access to the active site of the enzyme. We show that commercially available antiviral drugs which target HIV-1 protease can be divided into two classes, those which significantly affect flap rigidity and those which do not. The non-peptidic inhibitor tipranavir is distinctive in its consistently strong effect on flap rigidity. CONTACT: jack.heal@warwick.ac.uk; r.roemer@warwick.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rapid simulation of protein motion: merging flexibility, rigidity and normal mode analyses.

Protein function frequently involves conformational changes with large amplitude on timescales which are difficult and computationally expensive to access using molecular dynamics. In this paper, we report on the combination of three computationally inexpensive simulation methods--normal mode analysis using the elastic network model, rigidity analysis using the pebble game algorithm, and geometric simulation of protein motion--to explore conformational change along normal mode eigenvectors. Using a combination of ElNemo and First/Froda software, large-amplitude motions in proteins with hundreds or thousands of residues can be rapidly explored within minutes using desktop computing resources. We apply the method to a representative set of six proteins covering a range of sizes and structural characteristics and show that the method identifies specific types of motion in each case and determines their amplitude limits.

Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization.

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.

Comparative analysis of rigidity across protein families.

We present a comparative study in which 'pebble game' rigidity analysis is applied to multiple protein crystal structures, for each of six different protein families. We find that the main-chain rigidity of a protein structure at a given hydrogen bond energy cutoff is quite sensitive to small structural variations, and conclude that the hydrogen bond constraints in rigidity analysis should be chosen so as to form and test specific hypotheses about the rigidity of a particular protein. Our comparative approach highlights two different characteristic patterns ('sudden' or 'gradual') for protein rigidity loss as constraints are removed, in line with recent results on the rigidity transitions of glassy networks.

Viral neoplastic transformation of hamster pineal cells in vitro: retention of enzymatic function.

Cultures of hamster pineal tissue infected with certain oncogenic DNA viruses undergo neoplastic transformation and produce tumors when injected into homologous hosts. Hydroxyindole-O-methyl transferase, an enzyme found exclusively in the pineal gland, is present in low concentrations in transformed pineal cells in vitro and in larger amounts in tumors produced by the injected cells. This enzyme is not present in several nonpineal tissues similarly transformed.

Inherited medullary thyroid carcinoma: a final monoclonal mutation in one of multiple clones of susceptible cells.

Inherited medullary thyroid carcinomas contain one form of glucose-6-phosphate dehydrogenase (G6PD) in black female patients who are mosaic in normal tissues for G6PD types A and B. The same individual may have several tumors each containing either G6PD A or G6PD B. The data suggest that the inherited defect is an initial mutation producing multiple clones of defective cells; each tumor then arises as a final mutation in one clone of these cells.

Preservation of androgen secretion during estrogen suppression with aminoglutethimide in the treatment of metastatic breast carcinoma.

We evaluated the comparative effects of aminoglutethimide (AG) on androgen and estrogen levels estrone ([E1], estradiol [E2], plasma dehydroepiandrosterone-sulfate [DHEA-S], testosterone [T], dihydrotestosterone [DHT], delta 4-androstenedione [delta 4-A]), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin in postmenopausal patients with breast cancer randomly allocated to either AG treatment or bilateral surgical adrenalectomy as a control group. In response to either treatment, the plasma levels of E1 fell 62-75% (P less than 0.001) and urine E1 85.7-88.7% (P less than 0.001) in all study days over a 12-wk period. Similarly, the concentrations of E2 in plasma and urine fell 40-72% without statistically significant differences between the two treatment modalities. The relatively weak androgen, DHEA-S, was reduced by 92% (877.3 +/- 184.6 to 71.8 +/- 14.5 ng/ml) at 12 wk in women treated with AG, but suppressed nearly 99% (1,151 +/- 262 to 5.8 +/- 3.3 ng/ml) in adrenalectomized women. At all time points after treatment, the DHEA-S levels were significantly higher in patients receiving AG. Plasma concentrations of the potent androgens, T and DHT, were also relatively preserved during AG treatment. T levels were never significantly reduced by AG, and DHT concentrations were decreased only at the 4th wk to a maximum of 20%. delta 4-A levels fell 56% in response to this drug only on the 12th wk of therapy (basal, 0.79 +/- 0.09 ng/ml; 12 wk, 0.35 +/- 0.07 ng/ml). In marked contrast, all androgens fell significantly at each time period in response to surgical adrenalectomy, with an 81% maximum suppression of T, 73% of DHT, and 97% of delta 4-A. In response to estrogen suppression, plasma levels of FSH, LH, and prolactin did not change significantly throughout the treatment period in either therapy group. To examine possible contributions of the postmenopausal ovary to hormone levels during therapy, data from surgically castrate and spontaneously menopausal women were evaluated separately. No significant differences between the two groups were observed for E1, E2, T, DHT, DHEA-S, delta 4-A, LH, FSH, and prolactin. We conclude that equivalent and highly significant estrogen suppression occurs with either AG or surgical adrenalectomy although androgen secretion is preserved during AG treatment but not after surgical adrenalectomy. The combined effects of estrogen deprivation associated with androgen preservation might be significant in the therapeutic action of AG in hormone-responsive neoplasms.

RET gene and its implications for cancer.

The RET proto-oncogene encodes a protein receptor tyrosine kinase. RET mutations are associated with the dominantly inherited cancer syndromes multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC). In MEN 2A, MEN 2B, and FMTC, direct detection of RET mutations can be used to identify disease allele carriers prior to the development of clinically evident neoplasms. RET mutations are also associated with sporadic thyroid carcinomas. The effects of RET mutation on protein function have been investigated both in vivo and in vitro, and the study of RET has served to provide insights into the mechanisms of tumorigenesis in general.

Androgen stimulation of gross cystic disease fluid protein and carcinoembryonic antigen in patients with metastatic breast carcinoma.

Plasma levels of carcinoembryonic antigen (CEA) and the 15,000 molecular weight gross cystic disease fluid protein (GCDFP-15) were determined in 30 patients with metastatic breast carcinoma before, during, and after treatment with fluoxymesterone. Within 2 weeks after initiation of treatment, plasma levels of GCDFP-15 increased 50% above basal values in 15 (79%) of 19 patients. Similar increases in plasma CEA levels occurred in only 5 (23%) of 22 patients. Eight (33%) of 24 patients achieved increases in GCDFP-15 of 500% or more above basal levels after 14-336 days of therapy. Within 2 weeks of fluoxymesterone termination, 14 (93%) of 15 patients had a decrease in plasma GCDFP-15 levels, and in 12 (80%) the decrease exceeded 33% (the inverse of a 50% increase). Conversly, only 5 (33%) of 15 patients experienced a decrease in plasma CEA levels within 2 weeks of therapy termination, and in only 1 (6.7%) subject did the decrement exceed 33%. Nine (90%) of 10 patients who had 50% increases in plasma GCDFP-15 during initial androgen therapy also had significant decreases in plasma GCDFP-15 following termination of therapy. Data on 3 prospectively studied patients demonstrated that plasma GCDFP-15 rose within 24 hours of initiation of fluoxymesterone therapy and continued to rise for at least 6 days. Increased plasma levels of GCDFP-15 were reflected in increased urinary excretion of the glycoprotein.

Breast gross cystic disease fluid analysis. I. Isolation and radioimmunoassay for a major component protein.

Human breast gross cystic disease (GCD) fluid was analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, and four major proteins (GCDFP-70), GCDFP-44, GCDFP-24, and GCDFP-15) were identified. By fractionation techniques, these proteins were separated from one another. The GCDFP-70 was immunologically identical to human albumin and was present in GCD fluid at approximately a 100-fold lower concentration than in plasma. The GCDFP-44 was immunologically identical to human plasma Zn-alpha2-glycoprotein; however, it was present in GCD fluid at an approximately 50-fold higher concentration than in plasma. The GCDFP-24 was the major component protein of GCD fluid. It had progesterone binding activity, and immunologically it was identical to a component of human plasma; however, antisera that identified 30 separate components of plasma failed to identify the GCDFP-24 as one of these plasma proteins. The GCDFP-24 concentration in GCD fluid was approximately 100-fold higher than the plasma analog. The GCDFP-15 component was immunologically distinct from any plasma components, as judged by Ouchterlony analysis. It was, however, immunologically identical with a component of both human milk and saliva. As revealed by radioimmunoassay, plasma levels in normal subjects were 7-85 ng/ml. In patients with metastatic breast carcinoma, markedly plasma levels (150-30,000 ng/ml) of this protein were detected. Short-term tissue cultures of breast carcinoma explants released this protein into the culture medium.

Carcinoembryonic antigen in nonhuman primates.

Blood levels of carcinoembryonic antigen (CEA) have been measured in several nonhuman primate species. Only gorillas and chimpanzees were found to have significant elevations of CEA-like activity in their blood compared to the normal values of less than 2.5 ng/ml in humans. The average CEA level in 134 chimpanzees was 25.2 ng/ml (range, 4.2--95 ng/ml) and in 13 gorillas it was 32 ng/ml (range, 12.4--61.9 ng/ml). These levels were not related to sex. Blood levels repeatedly taken over a 1 1/2-year period remained relatively stable in both species. Analysis of parallelism of immunologic reactivity showed chimpanzee CEA to be similar to but not identical with human CEA. The molecular size of chimpanzee CEA was also similar to that of human CEA.

Antibody reacting with the murine mammary tumor virus in the serum of patients with breast carcinoma: a possible serological detection method for breast carcinoma.

Sera from patients with Stages A and B infiltrating ductal carcinoma of the breast, benign breast disease, cancers other than breast carcinoma, and normal female controls were examined by indirect immunoelectron microscopy (IEM) and a viral agglutination test for evidence of antibodies directed against murine mammary tumor virus (MMTV). Sera from 41 (79%) of 52 patients with breast carcinoma and eight (19%) of 42 normal subjects or patients with benign breast disease (noncancer subjects) showed evidence of MMTV labeling by IEM. In the MMTV agglutination test, significant virus agglutination (2+ to 4+) was present in eight (13%) of 61 noncancer sera, 58 (86%) of 68 breast carcinoma sera, and two (11%) of 18 other cancer sera. The results of the more rapid MMTV agglutination test correlated well with IEM. Analysis of reacting antibody by IEM revealed no immunoglobulin A and significant immunoglobulin M and immunoglobulin G antibody. Serum reactivity against MMTV was completely absorbed by MMTV but not by the glycoprotein with a molecular weight of 52,000 of MMTV, Friend murine leukemia virus, avian myeloblastosis virus, or sheep erythrocytes. It is concluded that reactivity of human antibodies to MMTV is strongly associated with, but is not entirely specific for, breast carcinoma. It remains to be determined if normal persons with these antibodies will ultimately develop breast cancer and should therefore be considered at high risk. These tests may have potential usefulness as a diagnostic screen for breast cancer.

Detection of the major glycoproteins of Friend leukemia virus (gp71) and the murine mammary tumor virus (gp52) on the surface of mouse cells.

Specific rabbit antisera to the major glycoproteins of Friend leukemia virus (gp71) and the mouse mammary tumor virus (gp52) were utilized to study the surfaces of C3H-, DBA-, BALB/c-, and C57BL-transformed and normal cells by immunoelectron microscopy. Antiserum to gp71 showed reactivity with all of the mouse cells tested regardless of strain, virus production, or state of transformation. In cells producing murine leukemia virus, budding viruses and other areas of the cell surface were consistently labeled with gp71 antiserum. A gp52-like antigen was likewise detected on both cell surfaces and virions of C3H and DBA cells producing the mammary tumor virus. Budding virions with surface spikes but with crescent-shaped nucleoids in C3H/HeJ cells were labeled specifically with gp52 antiserum. The antigen localized with anti-gp52 serum was detected in low concentration on the surface of nonvirus-producing cultures of a C57BL/6 sarcoma induced by the Schmidt-Ruppin D strain of Rous avian sarcoma virus (SRD-2), a BALB/c bone marrow culture (JLS-V9), and a normal BALB/c fibroblast culture (BALB/cF). Other cell cultures transformed by either C-type virus or methylcholanthrene failed to demonstrate gp52 antigen. Both gp52- and gp71-like antigens were found to be expressed simultaneously in C3H/HeJ, C3H-MT, DBA-MT, SRD-2 (transformed) and BALB/cF, JLS-V9, and C3H-1 (normal) cultures. Expression of gp52 antigen in the absence of gp71 was not detected in any of the cultures examined. These findings demonstrate the ubiquitous expression of gp71 in a wide variety of normal and transformed mouse cells while gp52 tends to be expressed predominantly in cells from mice with high mammary tumor incidence (C3H) and DBA), but only to a minor extent in cells from low mammary tumor incidence strains (BALB/c and C57BL).

Loss of heterozygosity of markers on chromosome 11 in tumors from patients with multiple endocrine neoplasia syndrome type 1.

Multiple endocrine neoplasia type 1 is an autosomal dominant condition characterized by the development of parathyroid hyperplasia, pituitary adenomas, and pancreatic islet cell tumors. Recently the gene for multiple endocrine neoplasia type 1 was mapped to the long arm of chromosome 11 between the loci PGA and INT2. We tested the hypothesis that tumor development is the result of a somatic deletion that unmasks a constitutional mutation. By investigating DNA isolated from tumors and somatic tissues in 12 patients from 4 different families with multiple endocrine neoplasia type 1, we found loss of heterozygous markers mapped to 11q13 in 9 (82%) of 11 informative tumors. In contrast, we were unable to identify allelic loss from other chromosomes using a variety of informative probes. This high incidence of chromosomal deletion of 11q13 suggests that this region is important in the oncogenesis of this disorder.

Low frequency of ras gene mutations in neuroblastomas, pheochromocytomas, and medullary thyroid cancers.

Little is known about the prevalence and significance of ras gene activation in neural crest tumors such as neuroblastomas, pheochromocytomas, and medullary thyroid cancers (MTCs). Therefore, we analyzed DNA from 10 human neuroblastoma cell lines and 10 primary human pheochromocytomas for activating mutations in N-ras, H-ras, and K-ras. We also studied DNA from 24 primary neuroblastomas and 10 MTCs for N-ras mutations. ras genes were analyzed by direct sequencing of specific DNA fragments amplified by the polymerase chain reaction. With the exception of the SK-N-SH cell line, the examined ras gene sequences were normal in all the neuroblastomas, pheochromocytomas, and MTCs tested. A single point mutation was identified at codon 59 (GCT(ala)----ACT(thr)) in one N-ras allele in an SK-N-SH subline. Interestingly, this mutation is different from the activating codon 61 mutation which resulted in the initial identification of N-ras from SK-N-SH DNA. Therefore, we analyzed the sequences of earlier passages and sublines of the SK-N-SH cell line, but mutations at codon 59 or 61 were not detected, suggesting that neither mutation was present in the primary tumor. Our results indicate that N-ras mutations may occur spontaneously during in vitro passage of cell lines but rarely, if ever, occur in primary neuroblastomas, pheochromocytomas, and MTCs. In addition, we have not found H-ras or K-ras mutations in any neuroblastoma cell line or primary pheochromocytoma.

Evaluation of chimpanzee antiserum to human carcinoembryonic antigen.

An adult chimpanzee (Pan troglodyte) with an endogenous circulating carcinoembryonic antigen (CEA) level of 60 ng/ml was immunized s.c. with human CEA. After 1 year of immunizations, the anti-human CEA antibody titer had plateaued. This chimpanzee antiserum demonstrated high avidity specific recognition of human CEA and showed ionic strength effects for CEA recognition similar to those previously described for goat and baboon anti-CEA antisera. Radioimmunoassay of 93 human plasma samples for CEA content using chimpanzee anti-CEA versus Roche goat anti-CEA antisera gave essentially identical results (R = 0.985). Endogenous CEA in chimpanzee blood was very poorly identified by chimpanzee anti-human CEA antisera compared to Roche goat antisera. Column chromatography of human and chimpanzee CEA in the presence of chimpanzee anti-CEA antibody showed only reactivity for the human CEA. In addition, chimpanzee antiserum had only minimal blocking effect on the binding of either goat or baboon antiserum to human CEA. We conclude from these studies that chimpanzee anti-human CEA antiserum recognized a determinant(s) on human CEA which was different from these recognized by goat or baboon antiserum to human CEA and this determinant(s) was poorly represented on chimpanzee CEA. In contrast, the human CEA determinant(s) recognized by baboon and goat anti-CEA antiserums were readily detected on chimpanzee (CEA).

Allelic loss on a chromosome 17 in ductal carcinoma in situ of the breast.

Multiple tumor suppressor genes are implicated in the oncogenesis and progression of invasive carcinoma of the breast. To investigate the chronology of genetic changes we studied loss of heterozygosity on chromosome 17 in ductal carcinoma in situ, a preinvasive breast cancer. A microdissection technique was used to separate tumor from normal stromal cells prior to DNA extraction and loss of heterozygosity was assayed mainly using simple sequence repeat polymorphism markers and the polymerase chain reaction. Loss of heterozygosity on 17p was observed in 8 of 28 tumors (29%) when compared with normal control DNA, whereas no loss was seen on 17q, suggesting that at least one locus on 17p is involved early in the development of breast cancer.

Consistent association of 1p loss of heterozygosity with pheochromocytomas from patients with multiple endocrine neoplasia type 2 syndromes.

Pheochromocytomas and medullary thyroid cancers (MTCs) are neuroendocrine tumors which arise sporadically or as part of the multiple endocrine neoplasia type 2 (MEN-2) hereditary syndromes. The most consistent molecular genetic abnormality which has been described in these tumors is loss of heterozygosity (LOH) of the short arm of chromosome 1 (1p). This finding is particularly interesting because the predisposition gene for the hereditary form of these tumors has been mapped to chromosome 10, but LOH on chromosome 10 in MEN-2 tumors is found rarely. We have used a battery of 1p DNA probes to elucidate the region of loss of 1p in 18 pheochromocytomas and 27 MTCs. Using restriction fragment length polymorphism analysis, we identified loss of all or a portion of 1p in 12 of 18 pheochromocytomas. 1p LOH was identified in nine of nine pheochromocytomas in MEN-2A and -2B patients, compared with only two of seven sporadic pheochromocytomas. We also found 1p LOH in one of two von Hippel-Lindau patients. LOH on 1p was noted in only three of 24 informative MTCs, and these were from patients with MEN-2A. In most of the pheochromocytomas, the entire short arm of chromosome 1p appears to have been lost; however, in three of the non-MEN pheochromocytomas and in three MEN-2A MTCs, the region of loss is smaller, allowing estimation of the smallest region of overlap. The combined data for MTCs and pheochromocytomas suggest that the smallest region of overlap of LOH is bounded by D1S15 (1pter-p22) and D1Z2 (1P36.3), excluding a region around MYCL (1p32). Although other regions of 1p should not be completely ruled out, the data suggest that this region may harbor a tumor suppressor gene or genes whose inactivation is important in the development of these tumors. Furthermore, the strong association between 1p LOH and the MEN-2 syndromes, especially in pheochromocytomas, suggests a relationship between the predisposition gene on chromosome 10 and the loss of the suppressor gene on 1p. Alternatively, other loci may be more important in sporadic disease.