Pyoderma gangrenosum (PG) is a rare, destructive inflammatory disease in which a painful infiltration of the skin breaks down to a make an enlarging ulcer. The ulcers are usually characteristic with elevated and undermined borders and may be solitary or multiple. PG may present in association with systemic disease, such as inflammatory intestinal diseases, polyarthritis, gammopathy and others. The predilection sites are the lower extremities, buttocks and abdomen, but any area of the body may be involved. The female lower genital tract may also be affected (1–3). Therefore, this entity should be known by gynecologists. We report a case of vulvovaginal PG. A 55-year-old woman was admitted to the hospital with spontaneous ulcerations of the vulva, vagina and the right gluteal region that had developed during the preceding week. The ulcerations developed rapidly as painful redness and infiltrations of the skin, followed by necrotization. She stated that “pieces of the skin fell off,” resulting in deep and very painful ulcerations. Her general condition was otherwise unaffected, including no symptoms of bowel disorder. She did not use any drugs, including nonsteroid antiinflammatory drugs (NSAIDs) Examination revealed five deep ulcers located in the gluteal region, the vaginal introitus (Fig. 1), the vaginal wall (Fig. 2) and the cervix, with undermined borders, varying in size from 1 to 7 cm in diameter. Pyoderma gangrenosum ulcers of the vulva (the left labium minus and the posterior commissure) and the right gluteal region at the time of diagnosis. Note the undermined borders. Pyoderma gangrenosum ulcers of the anterior vaginal wall at the time of diagnosis. Biopsies from the ulcer margin showed nonspecific changes with ulceration and neutrophilic infiltration without certain signs of vasculitis. Immunohistochemical examination did not detect deposits of immunoglobulin complexes, fibrin or complement. Blood and urinalysis were normal except for an elevated sedimentation rate (81 mm), C-reactive protein (CRP) (114 mg/L) and P-potassium (4.4 mmol/L) and P-creatinine (99 µmol/L). Bacterial culture from the ulcers grew colonies of Escherichia coli, probably of no pathological significance. All other investigations, including P-antinuclear antibodies, P-ANCA immunofluorescence test, the tumor markers CEA and CA125, smear and chest X-ray were negative. A diagnosis of PG was made and treatment with cyclosporin A (Sandimmun Neoral®) 5 mg/kg body weight daily was started immediately as the only therapy. The therapeutic response was remarkable, resulting in a significant decline in inflammation within the first 24 h. After 1 week of treatment, the patient felt much better and the ulcers were healing. After 1 month of therapy the ulcers were partly healed, without signs of inflammation. After 2 months the ulcers were completely healed, just leaving some scars and a defect in the left labium minus that was caused by the biopsies. The cyclosporin A dosage was reduced stepwise and terminated 3.5 months after onset of the disease. At the 6-month follow-up there were no signs or symptoms of recurrence. We report this case to increase the attention regarding PG as a possible diagnosis when dealing with spontaneous ulcerations of the female genital area. The diagnosis may be easy, provided one is aware of this condition. The most important differential diagnoses are infectious ulcers, especially herpes simplex, syphilis and ulcus molle (chancroid), carcinomas and Bechet's disease. The clinical appearance of the ulcerations, the acute onset and the pain, combined with the absence of signs and symptoms of an infectious condition, will usually be a sufficient basis for the diagnosis. Infectious disease should be ruled out by means of adequate microbiological and serological tests. Histological examination is nonspecific as for ulcers of any cause and typically shows neutrophilic inflammation, edema, and thrombosis of small- and medium-sized vessels at the base of the ulcer (4). There are no specific laboratory markers for PG. Correct diagnosis is important for early therapeutic intervention, and also for avoiding unnecessary surgical procedures that may easily increase the ulcerations, especially in the acute phase of the disease. Cyclosporin A is usually very effective in the treatment of PG (5). The disease may relapse after termination of the immunosuppressive therapy, requiring sustained treatment, possibly for several years. As PG is associated with inflammatory bowel disease, these patients should be queried specifically with respect to these conditions.

A full-length cDNA encoding human parathyroid hormone (hPTH) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 μg/l in the medium per 7.5 × 106 cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t‐antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS‐PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley‐Liss, Inc.

The entire human parathyroid hormone (hPTH) cDNA gene with its natural signal and pro-region is expressed in transfected mouse mammary tumor cells (C127I cells) and Chinese hamster lung cells (DON cells) under control of the murine metallothioneine-1 promoter in a vector in which replication functions are provided by the entire genome of bovine papilloma virus type I (BPV-1). Authentic hPTH is efficiently produced by the non-endocrine cells and secreted to the growth medium without any abberant processing. Immunoblots from SDS-PAGE gels of concentrated growth medium reveal one band corresponding to intact, undegraded hPTH. Purification by reversed-phase HPLC results in a peptide with an amino acid content and N-terminal sequence identical to hPTH. For comparison, hPTH cDNA with deleted prepro-region is also expressed as secretory proteins in Escherichia coli and in Saccharomyces cerevisiae. In E. coli the vector construct is based on the staphylococcal protein A promoter employing protein A signal sequence. In S. cerevisiae a mating factor α expression system containing the factor α-signal sequence is employed. The results show that intact hPTH is secreted in addition to proteolytically cleaved fragments in both microorganisms. Thus, the signal sequences promote efficient secretion, and correct N-terminal processing of hPTH in both mammalian, bacterial and yeast cells. However, the folding characteristics of hPTH make it susceptible to internal proteolytical cleavage which appears to be species specific in yeast and E. coli.

Two staphylococcal protein-A signal sequences were constructed and tested for function in Escherichia coli, after being linked to human parathyroid hormone (hPTH) cDNAS representing the intact form (1-84 amino acids) and two N-terminal (1-37 and 1-7 amino acids) peptides. One signal sequence was identical to the wild type, and the other signal contained a deletion of 12 bp at the 3' end. The truncated hPTH cDNAs were fused at their 3' ends to IgG-binding domains (ZZ) derived from protein A in order to facilitate purification and characterization. The expression plas mid pSPTH, containing the wild-type signal sequence, secreted efficiently the intact recombinant hPTH (1-84) into the medium. Plasmids containing the truncated hPTH genes after the wild-type signal, gave rise to hPTH-ZZ hybrid proteins which were correctly processed at the N-terminal, but the major fractions appeared in the periplasmic compartment. In contrast, the plasmid pS'PTH which harboured the 4-amino-acid signal deletion did not promote a uniform secretion of intact hPTH (1-84) to the medium, but released a non-processed form both into the periplasmic compartment and to the medium. The related plasmids pS'PTH37ZZ and pS'PTH7ZZ with the mutated signal sequence gave rise to small or trace amounts of unprocessed forms of fusion proteins in the medium and periplasm, thus the secretion competence was markedly reduced. Thus, for correct N-terminal processing, we conclude that the amino acid sequence in the signal adjacent to the expressed protein, is a key determinant. However, release into the medium or periplasmic space appeared to be dependent also on protein folding, irrespective of signal-sequence cleavage. Furthermore, we observed that the peptides with the wild-type signal sequence and correct N-terminal processing, were the only forms that showed internal cleavage of hPTH. Uncleaved signals may contribute to folding characteristics of the ensuing protein and e.g., prevent internal proteolysis.

The molecular structures of the receptors of growth hormone and prolactin have recently been identified by molecular cloning, and have been characterized in a number of species and different tissues. The receptors consist of one polypeptide chain with a single transmembrane region. The extracellular region contains cysteines for disulphide bonding and potential sites for glycosylations. The intracellular part which mediates the biological actions displays considerable heterogeneity of size. A high degree of homology is demonstrated between the extracellular part of these receptors and the receptors of a number of cytokines, interferons and growth factors. Together they comprise a new family of receptors called the cytokine/growth hormone/prolactinreceptor family. Their structure is reviewed, along with the actions of growth hormone and prolactin in vivo. In spite of numerous biological effects the molecular mechanisms of actions for this class of receptors are unknown, even though they convey important cell regulatory functions. The molecular analysis of growth hormone receptor has provided new medical insight on the results of growth hormone replacement therapy in persons with deficient growth.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1–37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1–37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.

Genetic parameters were estimated from data recorded at slaughter on 2–4-kg rainbow trout (Salmo gairdneri) from three year-classes. Observations were made on 4466 fish of 47 sires and 249 dams nested within sires (data set I). Some traits were recorded only on a sample of 662 fish of 37 sires and 111 dams included in data set I (data set II). No sire or dam was used in more than one year-class.Heritabilities based on the sire component of variance for traits in data set I were 0.21 for ungutted and gutted body weight, 0.18 for body length, 0.36 for dressing percentage, 0.19 for condition factor, 0.23 for gonad weight, 0.32 for belly thickness score and 0.40 for belly thickness measured, 0.25 for abdominal fat score and 0.28 for viscera weight, and 0.27 for meat colour score.Heritabilities for traits in data set II were 0.22–0.30 for body circumference, 0.19–0.34 for size and belly thickness measurements of the cross sections at the pelvic and dorsal fins, 0.13–0.25 for body shape, 0.00–0.31 for shape of the cross sections and 0.33, 0.47 and 0.03 for percent water, fat and protein in the meat, respectively.Genetic correlations of body weight with (a) body circumference traits and size measurements of the cross sections were all very high; (b) belly thickness, condition factor, abdominal fat score and meat colour score were low, but positive; (c) fat percentage was also low, but negative. Genetic correlations of belly thickness with abdominal fat score and fat percentage were −0.54 and 0.24, respectively, and −0.33 between fat percentage and abdominal fat score.Genetic correlations of condition factor with carcass fat percentage and body shape were 0.15 and 0.52, respectively, and the phenotypic correlations were 0.16 and 0.82, respectively. It was concluded that condition factor is an adequate measure of body shape.In conclusion, the possibilities for genetic improvement of body weight, belly thickness, body shape, abdominal fat, fat in the meat and meat colour are very good in rainbow trout. No strong unfavourable genetic correlation was found among the traits.

Using a microinjection method (Rokkones et al. 1985) deoxyribonucleic acid was introduced into fertilized salmonid eggs. The survival rate after a 28 day period was 91% for injected eggs in comparison to non-injected controls. A gene construct containing the mouse metallothionein promoter fused to the human growth hormone structural gene was microinjected either as a supercoiled plasmid or as a linear sequence. In Southern blot analysis of both 5 and 73 day old dissected rainbow trout embryos, as well as in 1 year old Atlantic salmon, the mouse metallothionein human growth hormone gene sequence was detected together with the chromosomal DNA when micro-injected as plasmid or as linear DNA. After digestion with Bam HI restriction endonuclease, the human growth hormone gene was excised from the high molecular weight DNA fraction. Transcription into human growth hormone specific RNA, as well as translation and release of human growth hormone immunoreactive protein, could be demonstrated in early embryonic stages.