AAV-P125A-endostatin and paclitaxel treatment increases endoreduplication in endothelial cells and inhibits metastasis of breast cancer.

Endostatin potentiates the antimitotic effects of paclitaxel (taxol) on endothelial cells (ECs). P125A-endostatin and taxol-treated ECs showed multipolar spindles and nuclear lobulation, leading to mitotic catastrophe and cell death. Induction of nuclear abnormalities was found to be dependent on ß-catenin levels as wnt-mediated overexpression of ß-catenin reversed the changes in nuclear morphology. These results prompted us to investigate whether antiangiogenic gene therapy and paclitaxel chemotherapy can synergistically inhibit angiogenesis and tumor growth. We first determined the effect of combination treatment in a transgenic mouse model of breast cancer. Intramuscular injection of recombinant adeno-associated virus type-2 virus induced sustained expression of P125A-endostatin. In vivo studies showed that combination therapy inhibited mammary cancer growth, delayed the onset of multifocal mammary adenocarcinomas, decreased tumor angiogenesis and increased survival in treated mice. In a second model, female athymic mice were orthotopically transplanted with a metastatic human breast cancer cell line. Antiangiogenic gene therapy in combination with paclitaxel inhibited tumor angiogenesis and lung/lymph-node metastasis in this model. These studies demonstrate cooperation between endostatin gene therapy and chemotherapy to inhibit tumor initiation, growth and metastasis.

Evaporation-induced self assembly of nanoparticles in non-buckling regime: volume fraction dependent packing.

Hierarchically structured micrometric spheres are synthesized by evaporation-induced self assembly of silica colloids using spray drying technique. Packing of nanoparticles during drying of droplets is an important issue. The motivation of the present work is to investigate the effects of concentration of initial colloidal dispersion on the packing of the nanoparticles in assembled grains in non-buckling regime of drying. It has been observed that the packing of nanoparticles inside the dried grains, even in the non-buckling regime, varies significantly with concentration. Although, the packing of nanoparticles remains uniform in an assembled grain at smaller concentration, the same becomes non-uniform at higher concentration. Further, the average packing fraction of the nanoparticles within the assembled grains, decreases with increasing colloidal concentration. These observations have been attributed to the modification in viscosity of the initial dispersion. Electron microscopy, light scattering measurements have been performed to probe overall morphology of the dried grains, while inter-particle correlation inside the grains has been investigated by small angle neutron scattering.

Buckling-driven morphological transformation of droplets of a mixed colloidal suspension during evaporation-induced self-assembly by spray drying.

Morphological transformation during evaporation-induced self-assembly of a mixed colloidal suspension in micrometric droplets has been investigated. It has been demonstrated that a buckling-driven shape transition of drying droplets of mixed colloidal suspension takes place during evaporation-induced self-assembly. Further, it is also shown that the distortion modulations get significantly amplified with enhancement in volume fraction of anisotropic soft colloidal component of the mixed colloids. It has been argued that the reduction in elastic modulus of formed shell, at the boundary of a drying droplet, and the anisotropic nature of one of the colloidal components facilitate the deformation process. Hierarchical structures of these assembled colloidal grains have been probed using electron microscopy and scattering techniques.

Sweat gland carcinoma - a case report.

A 54 years male presented with painless, indolent ulcers in the left axilla in an area approximately of 5 cm diameter. They were fixed to the underlying structures. Some lymph nodes eg, pectoral, apical and central groups were also enlarged. Incision biopsy for the margin of the ulcer was done. It showed adenocarcinoma of the sweat glands. Wide local incision with 2 cm margin of surrounding skin was performed. The patient was discharged after a course of adjuvant radiotherapy and is doing well.

Pulsations in the umbilical vein during labor are associated with increased risk of operative delivery for fetal distress.

OBJECTIVES: Under physiological conditions the blood flow velocity waveform in the umbilical vein (UV) has an even non-pulsating pattern. Pulsations in the UV have been described in human fetuses exposed to chronic hypoxia and heart failure. Current techniques for fetal surveillance during labor and delivery involve a risk of both over- and underestimation of fetal hypoxia. We aimed to examine whether pulsations in the UV appear in the human fetus during suspected intrapartum hypoxia, and if so whether they are associated with increased risk of operative delivery for fetal distress (ODFD). METHODS: This was a prospective double blind study including 52 normal pregnancies. A Doppler examination of the UV was performed on 26 fetuses with pathological and 26 fetuses with normal cardiotocography (CTG) during labor. Presence or absence of pulsations in the UV were noted and related to perinatal outcome. RESULTS: Pulsations in the UV were seen in eight (30.8%) of the fetuses with pathological CTG, of which six (75%) underwent ODFD. No pulsations were seen in the other 18 (69.2%) fetuses with pathological CTG and these were all delivered without ODFD. No pulsations were seen in the UV in the fetuses with normal CTG and these were all delivered without ODFD. Among the fetuses with pathological CTG, there was an increased risk of ODFD in fetuses with vs. those without pulsations in the UV (P < 0.0001). CONCLUSIONS: Pulsations in the UV can be observed in human fetuses during suspected intrapartum hypoxia and these pulsations are associated with an increased risk of ODFD. Doppler examination of the UV might give important additional information on fetal condition during labor and delivery.

Uterine and umbilical artery Doppler are comparable in predicting perinatal outcome of growth-restricted fetuses.

OBJECTIVE: To compare umbilical and uterine artery Doppler in predicting outcome of pregnancies suspected of fetal growth restriction (FGR). DESIGN: A prospective study included 353 singleton pregnancies complicated by an FGR fetus. SETTING: University Hospital setting. SAMPLE: Pregnancies suspected of FGR diagnosed by ultrasound fetal biometry during a 5-year period. MAIN OUTCOME MEASURE: Perinatal outcome in relation to uterine and umbilical artery Doppler. METHODS: The women underwent Doppler examination of the umbilical and uterine arteries. Results from the uterine, but not the umbilical artery, were blind to the woman and managing obstetrician. The Doppler results were related to perinatal outcomes including small for gestational age newborns, caesarean delivery, premature delivery (<37 weeks of gestation) and admission of the newborn to a neonatal intensive care unit. RESULTS: Abnormal uterine artery Doppler velocimetry was seen in 120 (33.4%) pregnancies and abnormal umbilical artery Doppler in 102 (28.4%). There was a statistically significant correlation between abnormal Doppler of both the umbilical and uterine arteries and adverse outcome of pregnancy. The two vessels were comparable in predicting adverse outcome. Women with normal umbilical artery Doppler (251) were analysed separately. Abnormal uterine artery Doppler, seen in 61 (24.3%) of those women, showed a statistically significant correlation for adverse outcome of pregnancy. CONCLUSIONS: Doppler examinations of the uterine and/or the umbilical arteries seem to be comparable as predictors of outcome in pregnancies complicated by FGR. Including uterine artery Doppler in the surveillance of growth-restricted fetuses might detect a group of pregnancies at high risk, even though the umbilical artery Doppler was normal.

Effect of microbial inoculation during vermicomposting of different organic substrates on microbial status and quantification and documentation of acid phosphatase.

In this experiment, three microbial strains were inoculated in two different organic wastes to study their effect on the humic acids content, acid phosphatase activity and microbial properties of the final stabilized products. Pyrophosphate extract of vermicomposts were analyzed through polyacrylamide gel electrophoresis to study the nature of a isozymes in different treatments. Results suggested that vermicomposting increased humic acids content and acid phosphatase activity in organic substrates and microbial inoculation further enhanced the rate of humification and enzyme activity. Although humic acids content in different microorganism-inoculated vermicomposts were statistically at par, acid phosphatase activity in these treatments was significantly (P<0.05) different. Results revealed that microbial respiration was increased due to vermicomposting, but a reduction in microbial biomass was recorded after stabilization of organic wastes. Although vermicomposting increased the value of microbial quotient (qCO(2)), microbial inoculation did not show any significant effect on qCO(2). The zymogram revealed that two isozymes of acid phosphatase (group II and group III) were present in all vermicompost samples and higher acid phosphatase activity in fungi-inoculated vermicomposts might be due to the presence of an additional isozyme (group I) of acid phosphatase.

Piggery wastewater treatment in a tropical climate: biological and chemical treatment options.

A novel biological treatment system was developed for the treatment of piggery wastewater under tropical conditions. It consisted of three consecutive sponge-based floating biofilters. The Upflow Anaerobic/Anoxic/Aerobic Floating Filter (UA3FF) system was shown to be effective with carbonaceous and, particularly, nitrogenous matter. The rationale for the processes occurring in anoxic-aerobic reactors was based on the concept of nitritation-denitritation rather than nitrification-denitrification. The N-related microbial communities manipulated by changing DO concentration and hydraulic retention time were able to effect a considerable increase in the total and specific N-removal (70% and 0.6 kg N m(-3) filter media per day, respectively) as compared to data reported elsewhere. Fluorescence In Situ Hybridization and Polymerase Chain Reaction amplification of ammonia monooxygenase (amoA) gene were used to study interrelationships between N-related microbial groups in the system. Microbiological data was interpreted in terms of operational behavior and performance of the reactors. The N-removal efficiency of the biological UA3FF system was compared with a combined biological/physicochemical system based on (a) biological anaerobic pretreatment followed by (b) a chemical precipitation (CP) and (c) an air stripping. Both systems were scrutinized as to operational advantages and costs. The treatment options could produce effluent of a high quality (202 mg COD l(-1), 126 total-N l(-1) and 89 mg COD l(-1) 48 total-N l(-1) in the biological and combined biological/physico-chemical treatment options, respectively) amenable for the subsequent treatment at the municipal facilities. However, the UA3FF biological treatment system was superior to the combined system by a factor of 20 as far as costs are concerned.

Changes in organic - C, N, P and K and enzyme activities in vermicompost of biodegradable organic wastes under liming and microbial inoculants.

The aim of this work was to study the effect of different organic wastes, viz. cow dung, grass, aquatic weeds and municipal solid waste with lime and microbial inoculants on chemical and biochemical properties of vermicompost. Cow dung was the best substrate for vermicomposting. Application of lime (5 g/kg) and inoculation of microorganisms increased the nutrient content in vermicompost and also phosphatases and urease activities. Bacillus polymyxa, the free-living N-fixer, increased N-content of vermicompost significantly (p < or = 0.01) as compared to other inoculants.

Transcriptional regulation via the NF-kappaB signaling module.

Stimulus-induced nuclear factor-kappaB (NF-kappaB) activity, the central mediator of inflammatory responses and immune function, comprises a family of dimeric transcription factors that regulate diverse gene expression programs consisting of hundreds of genes. A family of inhibitor of kappaB (IkappaB) proteins controls NF-kappaB DNA-binding activity and nuclear localization. IkappaB protein metabolism is intricately regulated through stimulus-induced degradation and feedback re-synthesis, which allows for dynamic control of NF-kappaB activity. This network of interactions has been termed the NF-kappaB signaling module. Here, we summarize the current understanding of the molecular structures and biochemical mechanisms that determine NF-kappaB dimer formation and the signal-processing characteristics of the signaling module. We identify NF-kappaB-kappaB site interaction specificities and dynamic control of NF-kappaB activity as mechanisms that generate specificity in transcriptional regulation. We discuss examples of gene regulation that illustrate how these mechanisms may interface with other transcription regulators and promoter-associated events, and how these mechanisms suggest regulatory principles for NF-kappaB-mediated gene activation.

Evaluation of third trimester uterine artery flow velocity indices in relationship to perinatal complications.

OBJECTIVE: Uterine artery Doppler is becoming a routine part of pregnancy surveillance in high-risk pregnancies. Which blood flow velocity waveform index to measure is debated and the 'notch' in early diastole is not widely accepted, as it is a subjective measure. The aim of the present study was to evaluate the different indices in the prediction of adverse outcome of pregnancies suspected for intrauterine fetal growth restriction (IUGR). METHODS: Uterine artery blood flow was recorded in 217 pregnancies admitted for Doppler ultrasound surveillance due to suspected IUGR. The median gestational age at examination was 38 weeks (range 25-42 weeks). Only cases having bilateral uterine artery notching were included in the evaluation. The uterine artery Doppler spectrum was analyzed for different indices, including evaluation of notch and end-diastolic velocities. Umbilical artery Doppler velocimetry was also performed. The outcome variables chosen were: a small-for-gestational-age (SGA) newborn, preterm birth, and abdominal delivery. ROC-curve calculations were used to compare the different indices. RESULTS: The uterine artery blood velocity pulsatility index (PI) and resistance indices (RI) were the best predictors of adverse outcome of pregnancy. Apart from premature birth, the systolic/end-diastolic ratio was less predictive of adverse outcome. The indices including only diastolic blood velocities were the least predictive of adverse outcome. The group with notch velocity above end-diastolic velocity was compared with those having notch velocity below the end-diastolic velocity. No difference in outcome was seen between the two groups. CONCLUSIONS: RI and PI as measures of third trimester utero-placental vascular impedance are the best predictors of adverse outcome of IUGR-suspected pregnancies.

Analysis of differentially expressed genes in hyperthyroid-induced hypertrophied heart by cDNA microarray.

Experiments were carried out to identify the altered genes in hyperthyroid rat heart and their influence on the functions of cardiac myocytes. Chronic treatment of rats with 3,5,3' triiodo-L-thyronine (T3) resulted in a prominent increase in the size of the left ventricle with increased wall thickness and reduced chamber volume leading to concentric cardiac hypertrophy. The heart weight to body weight ratio (HW/BW) in hyperthyroid rats was increased by about 58% over that of normal rats. Using cDNA microarray comprising 588 genes, we compared the differences in mRNA expression of hyperthyroid and normal rat heart. Based on a threshold of greater than 10% change, about 37 genes were found to be regulated by T3. Further analyses by Western blotting, Northern blotting and real-time quantitative RT-PCR of some of the genes confirmed the microarray results. The T3-altered genes encode various types of proteins related to metabolism, matrix and cytoskeletal structures, growth factors, transcription factors, Ca(2+)-channels etc. The physiological significance of one of these altered proteins in hyperthyroid heart, insulin-responsive glucose transporter (GLUT) type 4 (GLUT4), was studied in detail. The expression of GLUT4 was drastically reduced in the ventricular tissues of hyperthyroid heart. Insulin-induced glucose uptake in hyperthyroid cardiomyocytes was reduced significantly, indicating the impaired glucose transport in cardiac cells. Interestingly, a few genes such as GLUT4, cytochrome P450 isoforms, superoxide dismutase (SOD), collagens, matrix metalloproteinases (MMP), tissue inhibitors of matrix metalloproteinases etc. which had not been reported earlier were found to be altered in hyperthyroid heart. Our results show some new aspects of hyperthyroid heart which will be important in assessing the pathophysiology of hypertrophied cardiomyocytes.

Assessing the 'cyclodiode G-probe' using a grey scale test: reproducibility and differences between probes.

AIMS: 1. To determine the number of clinicians performing cyclodiode therapy who reuse the 'G-probe' used for the delivery of cyclodiode therapy. 2. To show a simple method to assess the output of the 'G-probe' that can be used in the clinical setting. METHODS: A total of 71 questionnaires were sent to ophthalmologists who have an Oculight SLx Iris Medical Diode Laser. Ophthalmologists were asked as to whether they performed cycloablative therapy using the 'G-probe' and whether they reused the G-probe. They were also asked as to the frequency of any reuse of probes. To determine the output of the 'G-probe', paper copies of a custom-made grey scale chart containing graded blocks of increasing shades of grey densities were produced. A special probe holder was made so that the G-probe tip could be held at a fixed distance from the grey scale chart. Laser burns were made on the grey scale using this arrangement and measurements of the burn size were made. After using 'standard settings' of 2000 ms and 2000 mW, 'threshold' burns were defined. Five new probes (with two different operators) were tested to assess the interprobe, interoperator, and intersheet variability of test. Probes were then tested for the burn size produced between 1000 and 3000 mW, and 1000 and 3000 ms. RESULTS: Results from the questionnaire showed that of the 44 respondents (62.0% response), 93.2% performed cyclodiode therapy with 58.5% reusing the G-probe. Among them, 56.1% reused probes on more than one occasion. Results from testing a new G-probe on the grey scale chart showed that with 'standard settings', highly reproducible burns at grey density 8 could be produced. No significant interprobe, interoperator, and intersheet variations were noted. Above 3 J of laser energy, the test could detect a 20% increase in energy settings and it was found that at levels of 4 J or above, alterations to the power setting had a greater influence on burn production than alterations to the time setting. CONCLUSIONS: This study demonstrates 1. that many clinicians in the UK reuse G-probes, 2. a simple, quick, and highly reproducible method to assess the laser output from the G-probe used for cyclodiode therapy. The method can help the ophthalmic surgeon to test the G-probe prior to commencement of therapy and with a standard treatment protocol, may produce a more predictable intraocular pressure reduction.

X-ray crystal structure of proto-oncogene product c-Rel bound to the CD28 response element of IL-2.

BACKGROUND: The proto-oncogene product c-Rel is a Rel/NF-kappaB family transcription factor that plays a critical role in lymphoid cell development and mediates CD28-induced expression of interleukin 2 (IL-2). The CD28 response element (CD28RE) in the IL-2 enhancer is nonameric and similar to the kappaB DNA target sites recognized by p65 homodimers. RESULTS: We have determined and refined the X-ray crystal structure of the c-Rel homodimer complexed to the CD28RE DNA site, 5'-AGAAATTCC-3', to 2.85 A resolution. The c-Rel homodimer binds CD28RE in a mode similar to that observed in the p65/IL-8 kappaB crystallographic complex. Binding studies reveal that the c-Rel homodimer recognizes the CD28RE with higher affinity as compared to other canonical kappaB sequences despite the nonconsensus A:T base pair at the 5' end of the CD28RE. Preferential recognition of the CD28RE by c-Rel results from the direct contacts between the protein and the DNA as well as intrasubunit interactions between the beta(f)-beta(g) loop in the dimerization domain and the DNA-contacting loop L1 of the N-terminal domain. Not only do these loops have different conformations in other Rel/DNA crystallographic complexes, but they also contain two of the five oncogenic point mutations found in v-Rel. CONCLUSIONS: The current structure indicates that a non-DNA-contacting loop in the dimerization domain and the DNA-contacting loop L1 may play critical roles in defining affinity and specificity. Two amino acid changes in these segments may account for the differential DNA binding by v-Rel as compared to that of c-Rel.

IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells.

NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.

Female sex pheromone of brinjal fruit and shoot borer, Leucinodes orbonalis blend optimization.

The brinjal fruit and shoot borer, Leucinodes orbonalis is the major pest of eggplant in South Asia. Analysis of female pheromone gland extracts prepared from insects of Indian and Taiwanese origin confirmed (E)-11-hexadecenyl acetate (E11-16:Ac) as the major pheromone component with 0.8 to 2.8% of the related (E)-11-hexadecen-1-ol (E11-16:OH), as previously reported from Sri Lanka. The average quantity of E11-16:Ac extracted per female was estimated to be 33 ng, with a range of 18.9 to 46.4 ng when collected 2 to 3 hr into the scotophase. In field trials conducted in India, blends containing between 1 and 10% E11-16:OH caught more male L. orbonalis than E11-16:Ac alone. At the 1,000 microg dose, on white rubber septa, addition of 1% E11-16:OH to E11-16:Ac was found to be more attractive to male L. orbonalis than either 0.1 or 10% E11-16:OH. Trap catch was found to be positively correlated with pheromone release rate, with the highest dose tested, 3,000 microg, on white rubber septa catching more male moths than lower doses. Field and wind tunnel release rate studies confirmed that E11-16:OH released from white rubber septa and polyethylene vials at approximately twice the rate of E11-16:Ac and that the release rate of both compounds was doubled in polyethylene vials compared to white rubber septa. This difference in release rate was reflected in field trials conducted in Bangladesh where polyethylene vial dispensers caught more male moths than either black or white rubber septa, each loaded with the same 100:1 blend of E11-16:Ac and E11-16:OH in a 3,000 microg loading.

The structure of Sky1p reveals a novel mechanism for constitutive activity.

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

Do locally agreed guidelines for optometrists concerning the referral of glaucoma suspects influence referral practice?

PURPOSE: To assess whether the issuing of local guidelines for glaucoma detection including a protocol for the referral of suspects can improve the quality and accuracy of referrals from optometrists. METHODS: Universally agreed guidelines, which included a protocol for referring glaucoma suspects, were circulated to all optometrists in the catchment area of a major teaching hospital ophthalmic unit. Data on 207 new patients referred to one glaucoma clinic were collected from GOS18 forms and hospital records covering two 12 month periods spanning the guideline dissemination. Referral accuracy was calculated and the reasons for protocol violations and referral of normal individuals were determined. Where possible, data were compared with a similar study relating to referrals made to the same clinic in 1988 and 1993. RESULTS: The mean (SD) intraocular pressure (IOP) at which optometrists referred patients was 22.5 (6.6) in 1997 and 21.9 mmHg (6.6) in 1998/9, both significantly less than in 1988. A statistically significant upward trend with time was found in the number of patients referred with an assessment of cup/disc ratio and in those with details of a visual field assessment. The overall 'true positive diagnosis' was 40% (42/105) in 1997 and 32.3% (33/102) in 1998/9 after the guidelines (p = 0.32), both of which were significantly lower than the 1988 rate of 56% (34/75) (p = 0.03 and 0.003). Fifty per cent of false positive referrals in both 1997 and 1998/9 were associated with an assessment by the optometrist of the optic disc(s) that was at variance with the ophthalmologist's. False positive visual fields were associated with 22% and 35% of non-true positive referrals in 1997 and 1998/9. Protocol violations were observed in 48% of referrals after the guidelines had been disseminated. When the referral was both a false positive and the guideline protocol was not followed, 88% of violations were associated with IOP measurement in 1997 and 73% in 1998/9. The equivalent figures for visual field violations were 70% in 1997 and 76% in 1998/9. Normal individuals referred as suspects were significantly (p = 0.001) less likely to be referred on IOP grounds if their optometrist followed the referral protocol. CONCLUSION: Local dissemination of glaucoma screening guidelines with a protocol for referral did not appear to improve the diagnostic accuracy of optometrists in our area. Optometrists who follow the guidelines refer fewer normal individuals on IOP grounds, but false positive visual fields and optic disc interpretation difficulties remain a factor in such referrals. Additional strategies will be necessary to improve the quality and accuracy of referrals for suspect glaucoma by optometrists.

Preparation and crystallization of dynamic NF-kappa B.Ikappa B complexes.

The formation of single, well-diffracting crystals is a requirement for any molecular structure determination by x-ray crystallography. Crystallization of biological macromolecules can represent a significant obstacle when the subject exhibits internal flexibility or indiscriminate self-association. In such cases, the removal of inherently flexible regions and the addition of stabilizing ligands can improve the probability of crystal formation and ordered growth. We have applied these principles in order to form crystals of the Rel homology region of transcription factor NF-kappaB in complex with its inhibitors IkappaBalpha and IkappaBbeta. None of these molecules crystallizes in the absence of a binding partner. Recombinant overexpression of truncated IkappaBalpha required selection of the correct start site. NF-kappaB.IkappaBalpha complex crystals formed under relatively stringent conditions. NF-kappaB. IkappaBbeta complex crystals were formed by analogy to NF-kappaB. IkappaBalpha, although some modifications in purification and complex formation were necessary due to differences between the inhibitors.