Shaping biological knowledge: applications in proteomics.

The central dogma of molecular biology has provided a meaningful principle for data integration in the field of genomics. In this context, integration reflects the known transitions from a chromosome to a protein sequence: transcription, intron splicing, exon assembly and translation. There is no such clear principle for integrating proteomics data, since the laws governing protein folding and interactivity are not quite understood. In our effort to bring together independent pieces of information relative to proteins in a biologically meaningful way, we assess the bias of bioinformatics resources and consequent approximations in the framework of small-scale studies. We analyse proteomics data while following both a data-driven (focus on proteins smaller than 10 kDa) and a hypothesis-driven (focus on whole bacterial proteomes) approach. These applications are potentially the source of specialized complements to classical biological ontologies.

Protein sequence annotation in the genome era: the annotation concept of SWISS-PROT+TREMBL.

SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation, a minimal level of redundancy and high level of integration with other databases. Ongoing genome sequencing projects have dramatically increased the number of protein sequences to be incorporated into SWISS-PROT. Since we do not want to dilute the quality standards of SWISS-PROT by incorporating sequences without proper sequence analysis and annotation, we cannot speed up the incorporation of new incoming data indefinitely. However, as we also want to make the sequences available as fast as possible, we introduced TREMBL (TRanslation of EMBL nucleotide sequence database), a supplement to SWISS-PROT. TREMBL consists of computer-annotated entries in SWISS-PROT format derived from the translation of all coding sequences (CDS) in the EMBL nucleotide sequence database, except for CDS already included in SWISS-PROT. While TREMBL is already of immense value, its computer-generated annotation does not match the quality of SWISS-PROTs. The main difference is in the protein functional information attached to sequences. With this in mind, we are dedicating substantial effort to develop and apply computer methods to enhance the functional information attached to TREMBL entries.

In vitro poly-(ADP-ribosyl)ation of chromatin proteins in the rat tapeworm, Hymenolepis diminuta.

1. (ADP-ribose)-transferase activity in crude chromatin of H. diminuta was demonstrated. 2. Chromatin proteins were ADP-ribosylated in vitro and selectively extracted. 60, 12 and 18% of the (ADP-ribose)n of chromatin proteins was associated with total histones, histone H1 and histone H2B, respectively. 3. The extent of oligo-(ADP-ribose) compared to total (ADP-ribose)n in the chromatin fraction, in the histone fraction, the histone H1 fraction and the histone H2B fraction was 45, 60, 26 and 49%, with an average chain length of 2.8, 2.1, 1.8 and 2.6, respectively. 4. Analysis of (ADP-ribosyl)n-ated proteins by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that histone H1, histone H2B and a 35 kDa non-histone protein were major (ADP-ribose)n acceptors.

Moxalactam for obstetric and gynecologic infections. In vitro and dose-finding studies.

Moxalactam (LY 127935), a "third-generation" beta-lactam antimicrobial, has been shown to have promising in vitro activity against a wide spectrum of pathogens similar to those isolated from women with pelvic infections. Pharmacodynamic studies have shown that its serum half life is longer than 2 hours, which permits less frequent dosing. The current investigation was carried out in two parts: In the first phase, the minimal inhibitory concentration of moxalactam against 519 clinical isolates was determined and compared to antimicrobials used in infections caused by these microbes. In vitro activity of moxalactam comparable to that of clindamycin was demonstrated against B. fragilis and other Bacteroides species. There was similar activity to penicillin G and clindamycin against anaerobic gram-positive cocci and activity superior to amikacin was demonstrated against Enterobacteriaceae. The second part of this investigation was a clinical one and 100 women with pelvic infections were given treatment with moxalactam. With an initial dose of 3 gm/day, women with posthysterectomy cellulitis and pelvic inflammatory disease did well. Women with pelvic infections following cesarean section responded less readily to this dose; however, when the initial dose was increased to 6 gm/day, a 91% cure rate was effected. The results of these investigations indicate that moxalactam is useful as a single-agent antimicrobial for treatment of polymicrobial female pelvic infection.

Cefoxitin for prophylaxis in premenopausal women undergoing vaginal hysterectomy.

To determine the unbiased incidence and types of postoperative infection and their alteration(s) by antimicrobial prophylaxis, a prospective double-blind study was performed using perioperative intramuscular cefoxitin or placebo given to premenopausal women undergoing vaginal hysterectomy at Parkland Memorial Hospital. The clinical and surgical profiles of the 2 groups of women were similar, but there were marked differences in their postoperative clinical courses. Only 8% of the 50 women given cefoxitin had major postoperative infection, compared to 57% of the 49 women given placebo (P < .001); this was associated with a 2.8-day reduction in the hospital stay for those given cefoxitin (P < .001). No clinically significant side effects were observed.

Cefamandole for treatment of obstetrical and gynecological infections.

Cefamandole nafate has been shown to have good in vitro activity against a wide spectrum of aerobic and anaerobic pathogens frequently isolated from women with obstetrical and gynecological infections. One hundred and twenty seven women with these infections were treated with cefamandole: 78 had post-cesarean section metritis; 24 acute pelvic inflammatory disease; 18 post-hysterectomy cuff cellulitis/abscess; and 7 had vulvar or abdominal wound abscesses. All but 13% of these women had either polymicrobial aerobic/anaerobic bacterial infections, or had an anaerobic infection alone. Of these 127 women, 116 responded to cefamandole administration alone, and in the other 11 chloramphenicol was added. Of these 11, surgical therapy was necessary to eradicate infection in six women. Phlebitis, mild to severe, was demonstrated in 14% of the women and responded to conservative measures. Of 402 bacterial isolates from these women, 94% were sensitive to cefamandole at 32 microgram/ml, an easily achievable serum level. Anaerobic streptococci were the most common isolate and 94% of these organisms were sensitive at 32 microgram/ml. Of the 43 Bacteroides species isolated, 90% were susceptible at 32 microgram/ml; 84% of Bacteroides fragilis were susceptible ast this concentration. Data now presented indicate that cefamandole given alone is safe and effective for treatment of women with polymicrobial mixed aerobic/anaerobic pelvic infections and approximately 5% will require surgical therapy for eradication of these infections.

Treatment of obstetric and gynecologic infections with cefamandole.

Cefamandole nafate is a derivative of 7-aminocephalosporanic acid which has been shown to have good in vitro activity against aerobes traditionally susceptible to cephalosporins as well as many anaerobes, including B. fragilis. One hundred women with obstetric or gynecologic infections completed treatment with cefamandole: 53 had post-cesarean section infections: 24, acute pelvic inflammatory disease: 16, posthysterectomy cuff cellulitis/abscess; and seven, vulvar or abdominal wound abscess. Almost 90% of these women had either polymicrobial aerobic/anaerobic bacterial infections or an anaerobic infection alone. Ninety women responded to cefamandole alone; in 10 cases chloramphenicol was added, but in addition five of these women required surgical therapy for eradication of infection. Mild to severe phlebitis at the infusion site that responded to conservative therapy was demonstrated in 14 women. Of 312 bacterial isolates from these women, 89% were sensitive to cefamandole at 32 microgram/ml, an easily achievable serum level; 93% of anaerobic streptococci, the most common isolates, were sensitive at 32 microgram/ml. Also, 90% of all Bacteroides species were susceptible at 32 microgram/ml; 82% of B. fragilis were susceptible at this concentration. These data indicate that cefamandole is safe and effective for treatment of women with polymicrobial pelvic infections but that approximately 5% of these women will require surgical exploration in addition to antimicrobial administration.

Infectious morbidity following cesarean section. Comparison of two treatment regimens.

During a 4-month period 265 women delivered by cesarean section were studied to determine what effect membrane rupture has on the incidence and severity of postoperative infection. There was a definite correlation between the duration of ruptured membranes and the incidence as well as severity of postoperative infections. Only 29% of women with intact membranes subsequently developed endometritis with pelvic cellulitis, in contrast to 85% of those whose membranes were ruptured for less than 6 hours. Wound and pelvic abscesses were encountered in less than 1% of women delivered with intact membranes, yet these complications developed in over 30% of women with membranes ruptured for less than 6 hours. The incidence of septicemia was four times greater in those women whose membranes were ruptured for less than 6 hours. Women with endometritis were treated with one of two empirical antimicrobial regimens chosen randomly. Intravenous penicillin and tetracycline was found to be as effective as, and perhaps slightly more effective than, the combination of intravenous penicillin and intramuscular tobramycin.

The bacterial pathogenesis of acute pelvic inflammatory disease.

To study the bacterial pathogenesis of acute pelvic inflammatory disease, peritoneal fluid was obtained by culdocentesis in 133 of 344 women with this disease. In 104 of the specimens bacteria were identified both in the gram-stained smear and culture. Neisseria gonorrhoeae was isolated from the lower genital tract in over half of these women, and there were 3 patterns of bacterial recovery from peritoneal fluid: N gonorrhoeae alone (22%), N gonorrhoeae and other organisms (32%), and nongonococcal organisms alone (46%). In women without cervical gonorrhea only nongonococcal organisms were identified from peritoneal fluid. In both groups of women a similar number of nongonococcal organisms were isolated. The results of this study supported those reported prior to availability of antimicrobials and suggest that N gonorrhoeae initiates most cases of pelvic inflammatory disease. A significant number of these women have superinfection with nongonococcal organisms which may preclude recovery of gonococci.

Evaluation of tetracycline or penicillin and ampicillin for treatment of acute pelvic inflammatory disease.

To evaluate guidelines for outpatient treatment of acute pelvic inflammatory disease recommended by the Center for Disease Control we studied 197 afflicted women. The women were treated either with tetracycline or with procaine penicillin and ampicillin, and 92% were subsequently seen at least once to assess efficacy of clinical and microbiologic treatment. Neisseria gonorrhoeae was isolated from the lower genital tract in 68% of these women, and although they had a quicker symptomatic response than those with nongonococcal infection (P less than 0.01), the two regimens were equally effective in producing clinical cure. However, subsequent identification of a pelvic abscess was 10 times more common in women from whom N. gonorrhoeae was not isolated. Therapy for pelvic inflammatory disease must be empirical since it is impossible to distinguish clinically between gonococcal and nongonococcal infection, and our data indicate that both regimens recommended by the Center for Disease Control are effective.