Birth weight for gestational age and the risk of infertility: a Danish cohort study.

STUDY QUESTION: Is birth weight for gestational age associated with infertility in adulthood among men and women? SUMMARY ANSWER: Being born small for gestational age (SGA) was associated with infertility in adulthood among men. WHAT IS KNOWN ALREADY: Fetal growth restriction may affect fertility, but results from previous studies have been inconsistent. STUDY DESIGN, SIZE, DURATION: In this population-based cohort study, we used data from a Danish birth cohort, including 5594 men and 5342 women born between 1984 and 1987. Information on infertility was obtained from Danish health registers during the period from the participants' 18th birthday and up until 31 December 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were men and women born in two Danish municipalities, Aalborg and Odense. Information on birth weight and gestational age was obtained from birth records, and information on infertility diagnoses and fertility treatment was retrieved from the Danish National Patient Registry (NPR) and the Danish In Vitro Fertilisation (IVF) registry. Information on potential maternal confounders was obtained from questionnaires during pregnancy and was included in adjusted analyses. Logistic regression analysis was used to estimate crude and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) for infertility according to birth weight for gestational age. MAIN RESULTS AND THE ROLE OF CHANCE: Men born SGA had a 55% higher risk of being diagnosed with or treated for infertility compared to men born appropriate for gestational age (AGA) (adjusted OR = 1.55, 95% CI: 1.09-2.21). The association attenuated after exclusion of men born with hypospadias or cryptorchidism (OR = 1.37, 95% CI: 0.93-2.01). No association was found between women's birth weight for gestational age and risk of infertility (adjusted OR = 1.00, 95% CI: 0.73-1.37). LIMITATIONS, REASONS FOR CAUTION: Estimation of gestational age is associated with some uncertainty and might have caused non-differential misclassification. The study design implicitly assumed similar distribution of reproductive and health-seeking behaviour across the groups that were compared. WIDER IMPLICATIONS OF THE FINDINGS: Men born SGA had a higher risk of infertility. Genital malformations may account for part of the observed association, but this must be explored further. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Health, Aarhus University. No competing interests are declared. TRIAL REGISTRATION NUMBER: N/A.

Sleep in critically ill, mechanically ventilated patients with severe sepsis or COPD.

BACKGROUND: The standard method for scoring polysomnographic (PSG) sleep is insufficient in the intensive care unit (ICU). A modified classification has been proposed, but has not been tested in specific groups of ICU patients. We aimed firstly to (1) use the modified classification to describe sleep in two groups of ICU patients: a severe sepsis group and a chronic obstructive pulmonary disease (COPD) group, and (2) to compare sleep stage distribution in the groups; secondly to compare the PSG findings with nurses' sleep evaluation. METHODS: Non-sedated mechanically ventilated patients with severe sepsis or COPD completed up to 20-hours PSG recording in each patient. A modified classification for scoring sleep in ICU was used for scoring the PSGs. Sleep assessment by nurses was done at 15 minutes intervals. RESULTS: We included 16 patients with severe sepsis and 17 patients with COPD. Half of the patients in the severe sepsis group and 59% in the COPD group had atypical sleep. We found significantly different sleep stage distribution in the two groups, with the COPD group having a higher proportion of atypical sleep (54.4% vs 48.7%, P < .0001). No correlation between nurse sleep assessment and PSG was found in cases of atypical sleep (P < .0001). CONCLUSION: Normal PSG sleep characteristics as defined by standard classification are absent in many conscious, non-sedated critically ill patients on mechanical ventilation. Nurse sleep evaluation does not correlate with PSG if atypical sleep is present in the PSG, which limits the reliability of subjective sleep assessment in this patient population.

Increasing BMI is associated with reduced expression of P-glycoprotein (ABCB1 gene) in the human brain with a stronger association in African Americans than Caucasians.

The efflux pump, p-glycoprotein, controls bioavailability and excretion of pharmaceutical compounds. In the blood-brain barrier, p-glycoprotein regulates the delivery of pharmaceutical substances to the brain, influencing efficacy and side effects for some drugs notably antipsychotics. Common side effects to antipsychotics include obesity and metabolic disease. Polymorphisms in the ABCB1 gene coding for p-glycoprotein are associated with more severe side effects to neuro-pharmaceuticals as well as weight gain, indicating a potential link between p-glycoprotein function and metabolic regulation. Using microarray data analysis from 145 neurologically sound adults, this study investigated the association between body mass index (BMI) and ABCB1 expression in the frontal cortex. Increasing BMI values were associated with a statistically significantly reduced expression of ABCB1. Investigation of DNA methylation patterns in a subgroup of 52 individuals found that the methylation/expression ratios of ABCB1 were unaffected by increasing BMI values. Interestingly, the effect of BMI on ABCB1 expression appeared stronger in African Americans than in Caucasians.

High BMI levels associate with reduced mRNA expression of IL10 and increased mRNA expression of iNOS (NOS2) in human frontal cortex.

Several studies link increasing body mass index (BMI) to cognitive decline both as a consequence of obesity per se and as a sequela of obesity-induced type 2 diabetes. Obese individuals are prone to a chronic low-grade inflammation as the metabolically active visceral fat produces proinflammatory cytokines. Animal studies indicate that these cytokines can cross the blood-brain barrier. Such crossover could potentially affect the immune system in the brain by inducing gene expression of proinflammatory genes. The relationship between obesity and neuroinflammation in the human brain is currently unknown. Therefore we aim to examine the relationship between BMI and gene expression of central inflammatory markers in the human frontal cortex. Microarray data of 141 neurologically and psychiatrically healthy individuals were obtained through the BrainCloud database. A simple linear regression analysis was performed with BMI as variable on data on IL10, IL1ß, IL6, PTGS2 (COX2) and NOS2 (iNOS). Increasing BMI is associated with a decrease in the mRNA expression of IL10 (P=0.014) and an increase in the expression of NOS2 (iNOS; P=0.040). Expressions of IL10 and NOS2 (iNOS) were negatively correlated (P<0.001). The expression of IL10 was mostly affected by individuals with BMI ⩾40. Multiple linear regression analyses with BMI, age, sex and race as variables were performed in order to identify potential confounders. In conclusion, increasing BMI could affect the IL10-mediated anti-inflammatory defense in the brain and induce iNOS-mediated inflammatory activity.

Effect of chronic antipsychotic treatment on striatal phosphodiesterase 10A levels: a [¹¹C]MP-10 PET rodent imaging study with ex vivo confirmation.

A number of phosphodiesterase 10A (PDE10) inhibitors are about to undergo clinical evaluation for their efficacy in treating schizophrenia. As phosphodiesterases are in the same signalling pathway as dopamine D2 receptors, it is possible that prior antipsychotic treatment could influence these enzyme systems in patients. Chronic, in contrast to acute, antipsychotic treatment has been reported to increase brain PDE10A levels in rodents. The aim of this study was to confirm these findings in a manner that can be translated to human imaging studies to understand its consequences. Positron emission tomography (PET) scanning was used to evaluate PDE10A enzyme availability, after chronic haloperidol administration, using a specific PDE10A ligand ([(11)C]MP-10). The binding of [(11)C]MP-10 in the striatum and the cerebellum was measured in rodents and a simplified reference tissue model (SRTM) with cerebellum as the reference region was used to determine the binding potential (BPND). In rats treated chronically with haloperidol (2 mg kg(-1) per day), there was no significant difference in PDE10A levels compared with the vehicle-treated group (BPND±s.d.: 3.57 ± 0.64 versus 2.86 ± 0.71). Following PET scans, ex vivo analysis of striatal brain tissue for PDE10A mRNA (Pde10a) and PDE10A enzyme activity showed no significant difference. Similarly, the PDE10A protein content determined by western blot analysis was similar between the two groups, contrary to an earlier finding. The results of the study indicate that prior exposure to antipsychotic medication in rodents does not alter PDE10A levels.

Regulation of the Bcas1 and Baiap3 transcripts in the subthalamic nucleus in mice recovering from MPTP toxicity.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure leads to significant and irreversible damage to dopaminergic neurons in both mice and humans. While MPTP exposure in humans causes permanent symptoms of Parkinson's disease, MPTP treated mice will recover behaviorally over a 3-week period. This mouse specific recovery might be linked to transcriptional changes in the basal ganglia enabling mice to maintain normal motor function in spite of low striatal dopamine levels. Laser microdissection was used to isolate the subthalamic nucleus from mice 7 and 28 days following MPTP exposure. High quality RNA was recovered and expressional analysis was performed on whole mouse genome microarrays. Identified regulated transcripts were validated in a separate batch of animals using quantitative PCR. Two transcripts with a significant regulation from days 7 to 28 in the MPTP treated groups, were identified: the brain specific angiogenesis inhibitor associated protein 3 (Baiap3) and the breast carcinoma amplified sequence 1 (Bcas1). Further studies of the molecular pathways involving these two transcripts may uncover processes in the subthalamic nucleus associated with the behavioral recovery observed after MPTP exposure.

Office systems intervention to improve diethylstilbestrol screening in managed care.

OBJECTIVE: To examine the effectiveness of a comprehensive office systems intervention for improving identification of diethylstilbestrol (DES) exposure, a low-incidence condition with potentially severe consequences. METHODS: We developed a comprehensive office systems intervention to facilitate screening and follow-up for women exposed to DES in utero, consisting of a DES toolkit and the clinical and administrative education necessary to use the tools effectively. The intervention was implemented in the internal medicine and obstetrics-gynecology departments at six free-standing health centers in a Boston-area staff-model health maintenance organization. Intervention sites were matched and paired with a comparison group of centers. Intervention effectiveness was assessed through pretest and posttest surveys of clinicians, medical record review of 3900 women, and review of a computerized medical records data base. RESULTS: There was significantly higher DES awareness and knowledge among clinical staff at intervention sites. Documentation of DES exposure in the medical record ranged from 1.14 to 2.31 times greater at intervention sites than in matched comparison sites, and rates of DES code use in pregnancy were 1.91 to 3.61 times greater. CONCLUSIONS: The office systems intervention improved documentation of DES exposure in a managed care environment. Because this approach was designed to accommodate the limited time allotted for each patient visit, it not only improved DES screening but could also serve as a model for integrating screening for other low-prevalence but potentially serious conditions into routine care.

Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions.

Here, we present a novel strategy for dissecting some of the steps involved in the squamous differentiation of the bladder urothelium leading to squamous cell carcinomas (SCCs). First, we used proteomic technologies and databases (http://biobase.dk/cgi-bin/celis) to reveal proteins that were expressed specifically by fresh normal urothelium and three SCCs showing no urothelial components. Thereafter, antibodies against some of the differentially expressed proteins as well as a few known keratinocyte markers were used to stain serial cryostat sections (immunowalking) of biopsies obtained from bladder cystectomies of two of the SCC-bearing patients (884-1 and 864-1). Because bladder cancer is a field disease, we surmised that the urothelium of these patients may exhibit a spectrum of abnormalities ranging from early metaplastic stages to invasive disease. Immunohistochemical analysis revealed three types of non-keratinizing metaplastic lesions (types 1-3) that did not express keratins 7, 8, 18, and 20 (expressed by normal urothelium) and could be distinguished based on their staining with keratin 19 antibodies. Type 1 lesions showed staining of all cell layers in the epithelium (with differences in the staining intensity of the basal compartment), whereas type 2 lesions exhibited mainly basal cell staining. Type 3 lesions did not stain with keratin 19 antibodies. In cystectomy 884-1, type 3 lesions exhibited the same immunophenotype as the SCC and may be regarded as precursors to the tumor. Basal cells in these lesions did not express keratin 13, suggesting that the tumor, which was also keratin 13 negative, may have arisen from the expansion of these cells. Similar results were observed with cystectomy 864-1, which showed carcinoma in situ of the SCC type. SCC 864-1 exhibited both keratin 19-negative and -positive cells, implying that the tumor arose from the expansion of the basal cell compartment of type 2 and 3 lesions. Besides providing with a novel strategy for revealing metaplastic lesions, our studies have shown that it is feasible to apply powerful proteomic technologies to the analysis of complex biological samples under conditions that are as close as possible to the in vivo situation.

A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis.

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).

Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities.

Fresh, superficial transitional cell carcinomas (TCCs) of low-grade atypia (3 grade I, Ta; 6 grade II, Ta), as well as primary cultures derived from them were labeled with [35S]methionine for 16 h, between 2 and 6 days after inoculation. Whole protein extracts were subjected to IEF (isoelectric focusing) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by autoradiography. Proteins were identified by a combination of proteomic technologies that included microsequencing, mass spectrometry, 2-D PAGE immunoblotting and comparison with the bladder TCC protein database available on the internet (http://biobase.dk/cgi-bin/celis). Comparison of the IEF 2-D gel protein profiles of fresh tumors and their primary cultures showed that the overall expression profiles were strikingly similar, although differing significantly in the levels of several proteins whose rate of synthesis was differentially regulated in at least 85% of the tumor/culture pairs as a result of the short-term culturing. Most of the proteins affected by culturing were upregulated and among them we identified components of the cytoskeleton (keratin 18, gelsolin and tropomyosin 3), a molecular chaperone (hsp 28), aldose reductase, GST pi, metastasin, synuclein, the calreticulin precursor and three polypeptides of unknown identity. Only four major proteins were downregulated, and these included two fatty acid-binding proteins (FABP:FABP5 and A-FABP) which are thought to play a role in growth control, the differentiation-associated keratin 20, and the calcium-binding protein annexin V. Proteins that were differentially regulated in only some of the cultured tumors included alpha-enolase, triosphosphate isomerase, members of the 14-3-3 family, hnRNPs F and H, PGDH, hsp (heat-shock protein) 60, BIP, the interleukin-1 receptor antagonist, the nucleolar protein B23, as well as several proteins of yet unknown identity. The suitability of in vitro bladder tumor culture models to study complex biological phenomena such as malignancy and invasion is discussed.

Analysis of chaperonin-containing TCP-1 subunits in the human keratinocyte two-dimensional protein database: further characterisation of antibodies to individual subunits.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research. CCT consists of at least eight different subunit types encoded by independent but related genes, and a set of antibodies that recognise individual subunits has proved useful in the characterisation and functional analysis of CCT. These antibodies were used to identify subunits of CCT in the human keratinocyte two-dimensional protein database. Accurate values for the pI and molecular mass of human CCT subunits were determined from the database, and biological data was obtained regarding changes in subunit levels in response to extracellular agents and growth conditions. The second part of the study describes the characterisation of seven monoclonal antibodies raised against mouse TCP-1, also known as CCT alpha, using a combination of epitope mapping and immunoblot analysis of protein extracts from different species and tissue types. Some antibodies were not monospecific for TCP-1, and a number of epitope-related proteins were identified.

Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas.

Multifocal recurrent papillary tumors provide a unique model system to study the molecular mechanisms underlying the steps involved in transitional cell carcinoma progression and offer a valuable source of material to search for biomarkers that may form the basis for diagnosis, prognosis, and treatment. We have examined the protein expression profiles of normal bladder urothelium and of 63 transitional cell carcinomas of various histopathological grades and T stages using high-resolution, two-dimensional gel electrophoresis, microsequencing, mass spectrometry, and a two-dimensional gel protein database approach for polypeptide identification (http://biobase.dk/cgi-bin/celis). In general, the results revealed a striking similarity between the overall qualitative expression patterns of papillary tumors of all grades, as well as of papillary and solid tumors of grade III. With few exceptions, tumors of grades I-III expressed, albeit at different levels, all of the keratins (7, 8, 13, 17, 18, 19, and 20) found in the normal urothelium. Grade IV tumors lacked or expressed reduced levels of keratin 13 but most resembled low-grade tumors. One invasive grade IV tumor, however, expressed a fibroblast-like protein phenotype. Four proteins that were expressed by normal urothelium and were lost at various stages of progression were identified as glutathione S-transferase mu, prostaglandin dehydrogenase (PGDH), a fatty acid binding protein with homology to the adipocyte isoform (A-FABP), and keratin 13. The percentage of tumors expressing A-FABP was very high in low-grade lesions but decreased drastically (P = 0.0006) in grade III and IV neoplasms. In addition, low-grade tumors contained more A-FABP than their high-grade counterparts. The stage of the disease was also statistically (P = 0.0269) related to the presence or absence of A-FABP in grade III tumors. Similar analysis of glutathione S-transferase mu and PGDH showed a statistically significant decrease of these proteins in high-grade (grades III and IV) tumors (P = 0.0026 and P = 0.0044, respectively). Only PGDH showed a suggestive correlation (P = 0.0775) with the stage of the disease in grade III tumors. Keratin 13 showed a drastic decrease in grade IV tumors. In addition to identifying biomarkers that may have prognostic value, our studies have suggested that A-FABP is an important component of the pathway(s) leading to bladder cancer development.

The master two-dimensional gel database of human AMA cell proteins: towards linking protein and genome sequence and mapping information (update 1991).

The master two-dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low-abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include "protein name", "antibody against protein", "cellular localization", and "microsequenced proteins". New entries include "human autoantigens" and "cDNAs". For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.

Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data.

A total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer-aided two-dimensional (2-D) gel electrophoresis. A master 2-D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988, 2,561-602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C-amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype-specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2-D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.

The MRC-5 human embryonal lung fibroblast two-dimensional gel cellular protein database: quantitative identification of polypeptides whose relative abundance differs between quiescent, proliferating and SV40 transformed cells.

A new version of the MRC-5 two-dimensional gel cellular protein database (Celis et al., Electrophoresis 1989, 10, 76-115) is presented. Gels were scanned with a Molecular Dynamics laser scanner and processed by the PDQUEST II software. A total of 1895 [35S]methionine-labeled cellular polypeptides (1323 with isoelectric focusing and 572 with nonequilibrium pH gradient electrophoresis) are recorded in this database, containing quantitative and qualitative data on the relative abundance of cellular proteins synthesized by quiescent, proliferating and SV40 transformed MRC-5 fibroblasts. Of the 592 proteins quantitated so far, the levels of 138 were up- or down-regulated (51 and 87, respectively) by two times or more in the transformed cells as compared to their normal proliferating counterparts, while only 14 behaved similarly in quiescent cells. Seven MRC-5 SV40 proteins, including plastin and two interferon-induced proteins, were not detected in the master MRC-5 images. The identity of 36 of the transformation-sensitive proteins whose levels are up or down regulated by two times or more was determined and additional information can be transferred from the master transformed human epithelial amnion cells (AMA) database (Celis et al., Electrophoresis 1990, 11, 989-1071) for those polypeptides of known and unknown identity that have been matched to AMA polypeptides. As more information is gathered in this and other laboratories, including data on oncogene proteins and transcription factors, this comprehensive database will outline an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways and cytoskeletal systems that may be directly or indirectly involved in properties associated with the transformed state.

A two-dimensional gel protein database of noncultured total normal human epidermal keratinocytes: identification of proteins strongly up-regulated in psoriatic epidermis.

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).

Identification of a group of proteins that are strongly up-regulated in total epidermal keratinocytes from psoriatic skin.

Analysis using two-dimensional (2D) gel electrophoresis of the [35S]-methionine-labelled proteins synthesized by non-cultured total epidermal keratinocytes obtained from normal and psoriatic skin revealed 6 proteins that are strongly up-regulated (5 times or more) in psoriatic skin. These proteins are synthesized at albeit lower levels by keratinocytes from normal and normal-appearing (uninvolved) skin of psoriatic patients, and correspond to isoelectric focusing sample spot numbers 4311 (40.3 kDa), 4003 (12.4 kDa), 5008 (11.9 kDa), 3012 (11.6 kDa), 6016 (11.6 kDa) and 1015 (10.1 kDa) in the normal keratinocyte 2D gel protein database [Celis et al, (1990) Electrophoresis, in press]. These proteins are also detected in the labelling medium indicating that they are at least in part secreted. Given their striking regulatory behavior, these proteins may play a role in the pathogenesis of psoriasis.

Two dimensional gel human protein databases offer a systematic approach to the study of cell proliferation and differentiation.

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to the advent of microsequencing the databases make it possible to directly link protein and DNA information.

Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels.

Coomassie blue-stained, heat-dried, and computer-imaged two-dimensional gels used to develop comprehensive human protein data bases served as the protein source to generate partial amino acid sequences. The protein spots were collected from multiple gels, rehydrated, concentrated by stacking into a new gel, electroblotted onto inert membranes, and in situ-digested with trypsin. Peptides eluting from the membranes were separated by HPLC and sequenced. Using this procedure, it was possible to generate partial sequences from 13 human proteins recorded in the amnion cell protein data base. Eight of these sequences matched those of proteins stored in data bases, demonstrating that a systematic analysis of proteins by computerized two-dimensional gel electrophoresis can be directly linked to protein microsequencing methods. The latter technique offers a unique opportunity to link information contained in protein data bases derived from the analysis of two-dimensional gels with forthcoming DNA sequence data on the human genome.