A semi-continuous method for purification of factor IX complex from human plasma.

We report the development of a semi-continuous method for preparation of factor IX complex from human plasma using ion exchange resins. Traditionally, stirred batch adsorption has been used due to the high pressure drops and low flow rates associated with soft gels in packed columns. Batch methods, however, typically involve higher labor costs and are more cumbersome in process environments. In the semi-continuous process, cryo-supernatant plasma is pumped through a 'stirred column' containing the resin. At both lab and pilot scale, higher recoveries of factor IX (FIX) were obtained at decreased total process times, compared to batch adsorption. A residence time of 15 min was found to be sufficient for capture of 95% of the FIX in the starting plasma. In the pilot plant, 550 liters of plasma was passed through a 50-liter column containing 8.5 liters of resin, yielding a 68% recovery of FIX. The results suggest that the recovery of FIX depends on the mode of contact (batch, continuous or packed column) between plasma and resin.

Reproductive senescence in female C57BL/6J mice: ovarian impairments and neuroendocrine impairments that are partially reversible and delayable by ovariectomy.

Ovarian and neuroendocrine impairments were examined before and after the age-correlated loss of estrous cycles in C57BL/6J female mice. The role of ovarian secretions in inducing neuroendocrine impairments before and after the loss of estrous cycles was also examined by determining neuroendocrine impairments after prolonged ovariectomy. Young (6-month-old) cycling, middle-aged (12-, 14-, and 16-month-old) cycling or acyclic, and old (18-month-old) acyclic mice were used. Ovarian impairments were assessed by grafting old ovaries into young hosts. Neuroendocrine impairments were assessed by grafting young ovaries into middle-aged and old hosts, ovariectomized either at grafting or 2 months before, and by inducing a LH surge in mice that had been ovariectomized for 4 days, 1 month, or 2 months. One group of 16-month-old and one group of 18-month-old mice were also ovariectomized at 6 months of age; these groups were used to examine the steroid-induced LH surge. The LH surge was induced by inserting sc a single priming estradiol (E2) implant, followed by two more (surge-inducing) implants 6 days later; 33 h after the second implantation, mice were decapitated (1800 h). The number of estrous cycles supported by middle-aged and old ovaries grafted into young hosts was reduced by more than 90% compared with that in young ovaries. The number of estrous cycles supported by middle-aged and old hosts given young ovarian grafts was reduced by 60% and 90%, respectively, compared with that in young hosts. Middle-aged and old hosts also had progressively longer estrous cycles than young hosts. Levels of the E2-induced LH surge in middle-aged and old mice were also reduced by 60% and 90%, respectively, compared with levels in young mice. In old acyclic mice, ovariectomy for 2 months partially reversed impairments in the LH surge and partially restored the ability to support cycles with young grafts; these functions, normally 90% impaired in old mice, were only 60% impaired after prolonged ovariectomy. Moreover, ovariectomy 2 months before grafting in old hosts resulted in shorter estrous cycles and 60% fewer pituitary adenomas. In middle-aged cycling mice, ovariectomy for 2 months did not affect impairments in the LH surge or in the ability to support estrous cycles with young grafts; these functions remained 60% impaired in middle-aged mice ovariectomized for 2 months. If mice were ovariectomized when young, the age-correlated impairments of the E2-induced LH surge at 16 months were largely prevented.(ABSTRACT TRUNCATED AT 400 WORDS)

Estradiol-induced adult anovulatory syndrome in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments.

Long-term effects of elevated plasma estradiol (E2) on ovarian and neuroendocrine functions were examined in 4-month-old cycling female C57BL/6J mice injected s.c. with 0.2 or 0.05 mg estradiol valerate (EV), or oil. Within 7 days, EV-injected mice became permanently acyclic, exhibiting the persistent vaginal cornification (PVC) characteristic of reproductive senescence in rodents. Four months after injection, ovaries from EV-injected mice exhibited no corpora lutea, but ovulated in response to an injection of human chorionic gonadotropin (hCG) (as do older, spontaneously PVC mice). When grafted into young mice, ovaries from EV-injected mice supported as many estrous cycles as ovaries from oil-injected controls. EV did not alter the suppression of luteinizing hormone (LH) by E2, LH response to injected LH releasing hormone (LHRH), or plasma prolactin (Prl). However, EV-injected mice exhibited impairments in LH regulation similar to those seen in old, acyclic mice. Plasma LH 30 days after ovariectomy was 40% lower, and E2-induced LH surges were 60% lower, in EV-injected mice versus controls. Furthermore, EV-injected mice were unable to support estrous cycles given young ovarian grafts, in contrast to controls. Effects of sustained but physiological levels (15-20 pg/ml) of plasma E2, were examined in intact cycling mice given sham or E2 implants. Six weeks after implantation, the implants were removed; only 50% of the E2-implanted mice subsequently exhibited estrous cycles, compared with 100% of sham-implanted controls. Furthermore, those E2-implanted mice which did cycle had fewer cycles than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of luteinizing hormone and prolactin in C57BL/6J mice: effects of estradiol implant size, duration of ovariectomy, and aging.

The induction of a LH surge by estradiol (E2) implants was characterized in ovariectomized C57BL/6J mice. Various times after ovariectomy mice were given a priming E2 implant, followed by an LH surge-inducing E2 implant, and were sampled 30 h later at darkness. The magnitude of the E2-induced LH surge was influenced by the postovariectomy interval, sizes of the implants, and age. Mice ovariectomized for 30-60 days before E2 implantation displayed larger surges than those ovariectomized for 4 days. Priming implants yielding 10 pg E2/ml plasma permitted the subsequent induction of vigorous LH surges, whereas no LH surges were observed with slightly larger priming implants that yielded 15 pg E2/ml. The size of the surge-inducing implant was correlated with the size of the subsequent LH surge. However, regardless of implant size, aging mice (8 vs. 13 months old) had smaller LH surges. Sequential daily LH surges were not observed under any conditions, suggesting that mice differ from rats and hamsters in their regulation of LH by E2. Plasma PRL was slightly elevated in the afternoon just before the LH surge, but returned to basal levels during the LH surge, indicating an uncoupling of the LH and PRL surges. The two-stage E2 implantation protocol for inducing LH surges by physiological levels of E2 allows more detailed examination of the priming vs. surge-inducing effects of E2.

Aging and the regulation of luteinizing hormone in C57BL/6J mice: impaired elevations after ovariectomy and spontaneous elevations at advanced ages.

The effects of age on the elevations of plasma luteinizing hormone (LH) after ovariectomy were studied in C57BL/6J mice. In longitudinal studies, mice ovariectomized at 4-6 months and sampled serially reached maximum plasma LH (200-300 ng/ml) after 1 month; these elevations were maintained to at least 15 months of age, when most intact mice have ceased cycling and have persistent vaginal cornification (PVC). In contrast, mice ovariectomized at 15 or 21 months had smaller LH elevations (to 150 ng/ml) 1 or 2 months after ovariectomy, as compared to younger (5- or 11-month-old) cycling mice whose LH was ca. 200 ng/ml 1 month after ovariectomy. The maintenance of LH elevations in chronically ovariectomized mice suggests that the smaller LH elevations in acutely ovariectomized mice of the same age may be an ovary-dependent aging phenomenon. Pilot studies detected no age effects on plasma LH turnover. Intact 20- to 26-month-old mice with leukocytic vaginal smears, low plasma estradiol (E2) and low progesterone had initially elevated plasma LH (ca. 125 ng/ml), if gross pathologic lesions were not present at necropsy. In contrast, old mice with cornified vaginal smears had higher plasma E2 and low (normal) plasma LH. The low plasma progesterone and elevated LH in older mice with leukocytic smears argues against a pseudopregnant status. Old mice of all vaginal smear types with pathologic lesions had low LH. Ovariectomy did not yield further increases of LH in old mice. The spontaneous elevation of plasma LH to ovariectomized levels in old intact mice with leukocytic vaginal smears and reduced plasma E2 implies that C57BL/6J mice eventually undergo ovarian changes analogous to menopause in humans.

Isolation and sequence determination of two pyridoxal 5'-phosphate-labeled thermolysin peptides from pig muscle phosphoglucose isomerase.

Pig muscle phosphoglucose isomerase modified with pyridoxal 5'-phosphate under conditions that cause at least 90% inactivation of its catalytic activity was found to incorporate about 1.5 eq of pyridoxal 5'-phosphate per subunit. After digestion with thermolysin, two pyridoxal 5'-phosphate-containing peptides were isolated and their amino acid sequences were determined to be Leu-Gly-pyridoxyl-Lys-Gln and Ile-Ala-Ser-pyridoxyl-Lys-Thr.

Large scale isolation of pig muscle phosphoglucose isomerase.

A large-scale purification procedure for phosphoglucose isomerase from pig skeletal muscle is described. It consists of two fractionations by selective precipitation and two ion exchange chromatography steps yielding an end product of approximately 900 units (micromoles of substrate converted to product per min per mg of protein, at 30 degrees) specific activity. The method separates three isoenzymic forms with an overall recovery of about 30% of the original total enzyme activity in the form of Isoenzyme III, the latter being the predominant enzyme species.