Subclinical cervicovaginal human papillomavirus infections associated with cervical condylomata and dysplasia. Treatment outcomes.

Seventy-seven women with subclinical cervicovaginal human papillomavirus (HPV) changes associated with cervical condylomata and/or mild or moderate cervical dysplasia were studied. All patients were treated with the CO2 laser for cervical lesions (condylomata and/or dysplasia) and, subsequently, based on their contraceptive history, were treated either with 5-fluorouracil (5-FU) cream or trichloroacetic acid (TCA). Those who did not wish to be treated were followed similarly to those who were treated. Overall there was no significant difference between those who had no treatment and those who received TCA or 5-FU. Those treated with 5-FU did more poorly as compared to the other two treatment groups with cervical condylomata and dysplasia at the three- and six-month follow-up (P < .05), and the results for TCA were not different from those in women who were observed and underwent no treatment. It appears that the treatment modalities used in this study did not have any beneficial effect on associated subclinical HPV infections.

Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami.

Two peptides with in vitro prolactin release-inhibiting activity were purified from stalk median eminence (SME) fragments of 20,000 pig hypothalami. Monolayer cultures of rat anterior pituitary cells were incubated with aliquots of chromatographic fractions and the inhibition of release of prolactin in vitro was measured by RIA in order to monitor the purification. The hypothalamic tissue extract was separated into 11 fractions by high-performance aqueous size-exclusion chromatography with one fraction showing a 4-fold increase in prolactin release-inhibiting factor (PIF) activity. This material was further purified by semipreparative reversed-phase (RP) HPLC. This process resulted in the separation of two distinct fractions that showed high PIF activity. These were further purified by semipreparative and analytical RP-HPLC to apparent homogeneity as judged by the UV absorbance profiles. Neither of the two peptides showed cross-reactivity with gonadotropin releasing hormone-associated peptide or with somatostatin-14 antibodies. Protein sequence analysis revealed that one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu- Ala-Cys - Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 of the N-terminal region of the proopiomelanocortin (POMC) precursor (corresponding to amino acids 1-26 of the 16-kDa fragment). The sequence of the other PIF was Ala-Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu- Leu-Arg - Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val- Tyr, representing residues 109-147 of the vasopressin-neurophysin precursor. Synthetic peptides corresponding to the N-terminal region of POMC had significant PIF activity in vitro.

Biological detectors in liquid chromatography.

The applicability of isolated organ preparations as chromatographic detectors was demonstrated. An appropriately designed detector cell (biological detector) applicable in both on-line and off-line modes was developed. Deproteinized sera from healthy volunteers and schizophrenic patients and human amniotic fluid were fractionated by gel-permeation, ion-exchange and reversed-phase liquid chromatography. Ultraviolet absorption and biological activity were compared. The results show that isolated organ preparations selected according to the needs of particular experiments meet the essential criteria of conventional chromatographic detectors. The use of isolated organs allows the detection of biologically active substances in a matrix of physicochemically closely related, but biologically distinct, fluid components. Biological detectors may also provide valuable additional information concerning the chemical structure of biologically active agents in an early stage of isolation.

Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.

To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically.

Method for isolation of MMP-7-ase isoenzymes from rat muscle tissue extract.

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HPLC columns. The procedures involved are extraction, ultracentrifugation, chromatographies and enzyme assays and require less than five hours.

Synthesis, purification and biological evaluation of porcine corticotropin-releasing factor.

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [Ile40]pCRF and [Asn40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [Ile40]pCRF and [Asn40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1-20 ng/ml. [Ile40]pCRF and [Asn40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [Ile40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn40]pCRF. The data indicate that synthetic porcine [Ile40]pCRF and [Asn40]pCRF have high biological activity.

Superactive octapeptide somatostatin analogs containing tryptophan at position 1.

We synthesized a series of octapeptide analogs of somatostatin, containing N-terminal tryptophan or another amino acid followed by the hexapeptide sequences Cys-Phe-D-Trp-Lys-Thr-Cys or Cys-Tyr-D-Trp-Lys-Val-Cys and a C-terminal threoninamide or tryptophanamide. After purification by HPLC, the inhibitory activities of these analogs on the release of growth hormone (somatotropin) in rats were determined in vivo. The eight octapeptides with an N-terminal tryptophan residue were found to have a greater inhibitory effect than somatostatin. The most potent of these analogs, D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2, was 94.3 times more active than somatostatin. The other analogs, in order of decreasing potency, were Ac-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2, D-Trp(For)-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2, D-Trp-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, Ac-Trp(For)-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2, Ac-Trp-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2, D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Trp-NH2, and D-Trp-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2. The growth hormone inhibitory activity of these analogs was from 53.7 to 11.6 times greater than that of somatostatin. The octapeptides containing D- or L-tryptophan at the N-terminus, phenylalanine at position 3, and threonine at position 6 exhibited a greater inhibitory effect on growth hormone release than that of the analogs with tyrosine and valine at positions 3 and 6, respectively. Substitution of D-tryptophan for D-phenylalanine at the N-terminus in the octapeptide containing phenylalanine in the third, threonine in the sixth, and threoninamide in the C-terminal position also increased the growth hormone-release inhibitory activity. Time-course assay showed that D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2 (RC-98-I), in a dose of 1 microgram/kg of body weight, inhibited the release of growth hormone for at least 3 hr. In view of their high activity and prolonged duration of action, some of these analogs could be useful clinically.

Data throwing more light on the causes of postpill amenorrhoea.

PIP: Post-pill amenorrhea was examined in a series of 64 Hungarian women by determining their serum levels of LH, FSH, prolactin, testosterone, dehydroepiandrosterone sulfate, estradiol and cortisol by radioimmunoassay. Subjects had taken the pills Infecundin, Ovidon, Bisecurin, Rigevidon, or Lyndiol for a least 6 months and had been amenorrheic at least 6 months. They were classified into groups as follows: hyperprolactinemic, hyperandrogenic, early menopausal, hypothalamic regulation disorder, and low-normal estradiol. Overweight was most often associated with hyperprolactinemia, hypothalamic disorder and hyperandrogenic women. LH was highest in the hyperandrogenic and hypothalamic groups. FSH was highest in ovarian insufficiency (p0.01). Free testosterone was low in hyperprolactinemia. Dehydroepiandrosterone was highest in hyperprolactinemia, reflecting adrenal function. Cortisol levels did not differ significantly. The results pointed to the fact that post-pill amenorrhea is not a single clinical entity, but a collection of individual disorders.^ieng

Clomiphene citrate response is predictable in corpus luteum insufficiency.

In order to predict the correct dose of clomiphene citrate in corpus luteum insufficiency, the results of the treatment of 117 patients were evaluated. Serum progesterone and prolactin concentrations were determined by RIA. A 3-fold increase in serum progesterone concentration was found following the 100 mg per day clomiphene citrate treatment, independently of the pretreatment progesterone or prolactin values. This means that in a well-selected, hormonally homogeneous group of patients, contrary to earlier observations, the clomiphene citrate sensitivity of the patients is predictable.

Endocrine consequences of danazol treatment in menorrhagia.

In order to study the endocrine side-effects of danazol, 15 patients with severe, benign menorrhagias, were individually treated with this drug to stop bleeding. LH, FSH, testosterone, free testosterone, DES-S, androstenedione, prolactin, estradiol and cortisol serum concentrations were measured before and after the 3 month treatment period. Danazol was found to be effective in reducing the amount of bleeding in patients with benign, severe menorrhagias; however, the weight gain of patients was significant during the 3 month period. LH and estradiol concentrations decreased; testosterone, free testosterone and dehydroepiandrosterone sulfate increased; prolactin, FSH, androstenedione and cortisol serum concentrations did not change, and SHBG disappeared from the serum, during the treatment period. We are of the opinion that danazol is an effective drug in menorrhagias with side-effects. In our view the hyperandrogenicity is the most important side-effect of this substance, which might be the result of some metabolites, but which might be the effect of a peripheral action of the drug as well.

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro.

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.

LHRH treatment of hypothalamic amenorrhoea.

Chronic intermittent intravenous administration of 13 micrograms LHRH every 88 minutes for 28 days, was successful in inducing ovulation in two patients with 3a type secondary hypothalamic amenorrhoea. A catheter phlebitis was the only side effect of the treatment and it could be easily prevented by more flexible, thin venous catheters. The minimum laboratory need for monitoring the treatment, and the absence of hyperstimulation of the ovaries suggested the superiority of this substance to hMG for ovulation induction in clomiphene negative patients.

PIP: Pulsatile LHRH administration is a practical approach for ovulation induction in clomiphene resistant patients with hypothalamic amenorrhea. LHRH (synthetic GnRH) is used to differentiate various types of secondary amenorrhea of functional origin. Immature (3c), prepubertal (3b), and adult (3a) amenorrheas have been differentiated by the negative gestagen test. 2 features make GnRH useful in ovulation induction: its ability to exert a self-priming function, thereby enhancing LH and FSH release, and its ability to modulate pituitary responsiveness and prevent ovarian hyperstimulation. 2 patients with hypothalamic amenorrhea were given LHRH administered intravenously, in a chronic pulsatile fashion. A portable computerized infusion pump, activated once every 88 minutes, delivered 13 mc of LHRH solution via an indwelling antecubital catheter. Blood samples were drawn daily or every other day, and plasma, LH, FSH, E2, and progesterone were measured by radioimmunoassay. Basal body temperature was also monitored. E2, LH, and FSH levels verified ovulation, and progesterone values showed normal corpus luteum function. Both patients showed a biphasic pattern of basal body temperature. Catheter phlebitis was the only side effect, which could be prevented by using a thinner, flexible catheter. The benefits of LHRH administration for ovulation induction are: no ovarian hyperstimulation; minimum laboratory monitoring requirements; and only the basal body temperature must be registered. These benefits suggest the superiority of LHRH administration over hMG administration for ovulation induction in clomiphene-resistant, normoadrenergic patients.

[Dynamics of exogenous progesterone in the blood of pigs stressed with polychlorobiphenyls (PCB)]. / Dynamika exogénneho progesterónu v krvi prasiat zatazených polychlórovanými bifenylmi (PCB).

For a period of 70 days, young gilts were given rations containing a commercial mixture of polychlorinated biphenyls (PCB), produced in Czechoslovakia (Delor 105). One group of animals (A) was given feed containing 10 mg chlorobiphenyls per kg, the other group (B) was given feed containing 50 mg chlorobiphenyls per kg; on the whole, each animal of group A got 1 g of PCB mixture and each animal of group B 5 g. In about the mid-time of the experiment and before its termination the animals were given an i. m. injection of progesterone; then the animals were studied for the changes in the concentration of blood plasma progesterone. Statistically significant (P less than 0.05) and highly significant (P less than 0.01) differences in the values of the biological half-life of progesterone were found between the group of PCB-treated animals and the control group, and between groups A and B.

Biochemical characterization of substances with myometrium contraction inhibitory action, originated from ovaries of pregnant sows.

In order to understand life itself, we need biotechnology to purify biologically active substances as well. One of these processes results peptides with myometrium contraction inhibitory effect, originated from the ovaries of pregnant sows, but different from relaxin. After collection and acidic-acetone extraction of the ovaries (product generation, primary purification) the substance with myometrium contraction inhibitory effect was further purified on Sephadex G-25 (fine), CM-52 and again Sephadex G-25 (fine) gels (secondary purification) without loosing the bioactivity of the substance (quality control). Product analysis by analytical high performance liquid chromatography and capillary isotachophoresis were performed at this stage of the experiments. The analysis mentioned above resulted in 2-15 different subfractions in the studied extract. This shows that the biologically active ovarian extract needs further purification before the planned final amino-acid analysis is performed.

Porcine follicular fluid treatment at proestrus diminishes the serum progesterone level of rats in early pregnancy.

Proestrous rats were treated with porcine follicular fluid (pFF) or porcine serum (pS) extract, afterwards they were put with males together. Next morning, the sperm positive females were considered as day 1 pregnant animals. On days 2, 8 and 14 of pregnancy serum progesterone level was determined by RIA. On days 2 and 8 of pregnancy serum progesterone level of pFF treated animals was significantly lower than that of pS treated ones, but it was not different from the controls on day 14 of pregnancy. The decreased progesterone level indicates that there are biologically active endogenous substances in the pFF (presumably inhibin or granulosa cell luteinization inhibitor) which may responsible for some forms of corpus luteum insufficiency and for some unexplained infertility cases.

[The effect of polychlorinated biphenyls (PCB) on liver aniline hydroxylase activity and on certain metabolic parameters in the blood of pigs]. / Vplyv polychlórovaných bifenylov (PCB) na aktivitu pecenovej anilínhydroxylázy a na niektoré metabolické parametre krvi prasiat.

For 70 days two groups of 10-week gilts were given feed supplemented with PCB commercial mixture of Czechoslovak make (Delor 105), at the doses of 10 and 50 mg. .kg-1 of feed. Over the experimental period, animals were administered 1000 mg or 5000 mg of PCB mixture. During the experimental period the levels of glucose, cholesterol, urea, total protein, vitamin A, calcium, inorganic phosphorus and the activities of AF, ALT and AST enzymes were determined twice. At the end of this experiment the aniline hydroxylase activity in microsomal fractions of liver was determined and pronounced induction effect of PCB on the enzymatic system was observed. Out of the studied metabolic parameters of blood plasma, the decrease in vitamin A level was significant.