A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.

A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.

Decreased histamine release by luteinizing hormone-releasing hormone antagonists obtained upon translocation of the cationic amino acid from position 8 to position 7.

We report analogues of N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Ilys-Pro-D-Ala- NH2, the parent antagonist (PA), which is a potent antagonist of LHRH. To simplify future radioactive labeling we prepared N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Arg-Pro-D-Ala-NH2 (4), [Arg8]PA, which had good activity in the antiovulatory assay (AOA). Other analogues were designed at first by substituting with Arg at positions 5, 6, 7, 9, and 10, and Trp or Leu at position 8. Subsequent analogues were prepared in attempts to improve the AOA of the initial ones. Substitutions with Arg9 or Arg10 led to analogues 1-3 with no AOA activity at 5 micrograms/rat. However, substitution with Arg7 gave 9, [Arg7,Leu8]PA, with significant activity in the AOA at 5 micrograms/rat and borderline activity at 2.5 micrograms/rat, and substitution with Ilys7 gave 13, [Ilys7,Leu8]PA, with borderline activity at 2 micrograms/rat, both analogues showing much weaker activity than PA in the histamine release assay (HRA) and therefore being potentially safer. Substitutions with D-Arg6 or Arg5 led to analogues with either good AO activity at 5 micrograms/rat (analogue 7) or with borderline activity at 5 micrograms/rat (analogue 8), although both were more potent than 6 in the HRA. Combinations of Ilys or Arg at positions 7 and 8 led to 10 and 11, both of which were tested at 2 micrograms/rat and found to have either good AO activity (analogue 10) or borderline activity (analogue 11) but unsuitably potent in HR. Substitutions using Ilys7 and neutral amino acids at position 8 led to 14-17 which were inactive in the AOA. Of great significance is the substitution with Arg7 yielding analogue 9, which was much safer in the HRA than analogue 4, [Arg8]PA. Analogues 9 and 13, featuring substitutions with the Arg7-Leu8 or Ilys7-Leu8 sequences were even safer than PA or 6 in the HRA. Analogue 12, [D-Trp3,Tyr5,D-Arg6,Arg7,Leu8]PA, featuring the Arg7-Leu8 sequence, had much lower potency in the HRA than [D-Trp3,Tyr5,D-Arg6,Leu7,Arg8]PA, which has the normal Leu7-Arg8 sequence. Ilys7 together with neutral amino acids at position 8 led to analogues 14-17 which were also very weak (safer) in the HRA, with the smaller amino acids Ala8 and Abu8 being the weakest of all analogues prepared.(ABSTRACT TRUNCATED AT 400 WORDS)

Design of potent oxytocin antagonists featuring D-tryptophan at position 2.

We prepared nine analogues (1-9) of MCPA-D-Phe-Phe-Ile-Asn-Cys-Pro-Arg-Gly-NH2, [MCPA1, D-Phe2, Phe3, Ile4, Arg8]oxytocin (MCPA = beta-mercapto-beta,beta-pentamethylenepropionic acid), a potent antagonist of the rat uterotonic action of oxytocin (OT). We replaced D-Phe with D-Trp and made [MCPA1,D-Trp2,Phe3,Ile4,Arg8]OT (1), which had OT pA2 of 7.51, somewhat higher than that of the D-Phe2 antagonist which has OT pA2 = 7.35 in our rat uterotonic assay. Both compounds are equipotent as antagonists of [Arg8]vasopressin in the rat antidiuretic assay, with pA2 = 8.1. Other substitutions gave [MCPA1,D-Trp2,4-Cl-Phe3,Ile4,Arg8]OT, (2), OT pA2 7.44; [MCPA1,D-Trp2,Phe3,Ile4,3,4-dehydro-Pro7,Arg8]OT (3), OT pA2 = 7.42; [MCPA1,D-Trp2,Phe3,Arg8]OT (4), OT pA2 = 7.58; [MCPA1,D-Trp2,Phe3,Arg8,Gly9-NHEt]OT (5), OT pA2 = 7.49; [MCPA1,D-Trp2,Ile4,Arg8]OT (6), OT pA2 = 7.46; [MCPA1,D-Trp2,Val4,Arg8]OT (7), OT pA2 = 7.58; [MCPA1,D-Trp2,Thr4,Arg8]OT (8), OT pA2 = 7.48; and finally, [MCPA1,D-Trp2,Arg8]OT (9), which was a more potent and more selective OT antagonist, with OT pA2 = 7.77 in the uterotonic assay and ADH pA2 less than 5.9 in the antidiuretic assay and hence is an important lead for the design of OT antagonists.

Improvement in potency of an oxytocin antagonist after systematic substitutions with L-tryptophan.

We report twelve analogues of [Pmp1,D-Trp2,Arg8]oxytocin, ANTAG (Pmp = beta, beta-pentamethylene-beta-mercaptopropionic acid), which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats, as measured in a uterotonic assay. Nine of the following analogues were designed by replacement of each of the nine residues in ANTAG with an L-tryptophan residue: [Ac-Trp1,D-Trp2,Val6,Arg8]OT, [Pmp1,Trp2,Arg8]OT, [Pmp1,D-Trp2,Trp3,Arg8]OT, [Pmp1,D-Trp2,Trp4,Arg8]OT, [Pmp1,D-Trp2,Trp5,Arg8]OT, [Aaa1,D-Trp2,Trp6,Arg8]OT, [Aaa1,D-Trp2,Val6,Arg8]OT, [Pmp1,D-Trp2,Ica7,Arg8]OT, [Pmp1,D-Trp2,Trp7,Arg8]OT, [Pmp1,D-Trp2,Trp8]OT, [Pmp1,D-Trp2,Arg8,Trp9]OT (11), [Pmp1,D-Trp2,Arg8,Trp(For)9]OT (12). In these analogues Aaa = 1-adamantaneacetic acid, and Ica = indoline-2-carboxylic acid. All linear analogues and analogues featuring Trp substitutions in the ring sequence of ANTAG were OT antagonists of lower potency than the parent peptide. All the analogues featuring Trp substitutions in the tail sequence of ANTAG were OT antagonists of equal or better potency than the parent peptide. Replacement with Ica7 gave analogue 8, equipotent with ANTAG, but replacement with Trp7 gave analogue 9, which shows almost a two-fold increase in potency (pA2 = 8.06). Replacement with Trp9 gave analogue 11 (pA2 = 8.03) which is about 1.8 times more potent than the parent antagonist, although Trp(For)9 had lower potency. Of great interest is that substitution with Trp8 leads to a more potent analogue, 10 (pA2 = 8.22), which, unlike most antidiuretic hormone antagonists, lacks any cationic charge in the molecule. The antidiuretic assay shows antagonists 9-11 to be weak antagonists of [Arg8]vasopressin, the antidiuretic hormone, with pA2 less than or equal to 6.0; hence, they may be interesting leads for future design of more potent and specific OT antagonists.

Haloperidol and nucleus accumbens dopamine depletion suppress lever pressing for food but increase free food consumption in a novel food choice procedure.

An important aspect of motivated behavior is that organisms will perform complex instrumental behaviors to gain access to stimuli such as food. In the present study, food-deprived rats were tested in an operant chamber in which the animals had a choice between pressing a lever to obtain a more-preferred food (Bioserve pellets), or free feeding on a less-preferred food (lab chow). Typically, rats pressed the lever to obtain the preferred food pellets, and ate little of the less-preferred food even though it was freely available. Pre-fed rats showed suppression of both lever pressing and feeding. Systemic administration of 0.1 mg/kg haloperidol (HP) led to a dramatic shift in the behavior of these rats, such that the number of lever presses was substantially reduced, but the amount of less-preferred food consumed showed a significant increase. This result occurred if the rats pressed a lever on either a CRF or FR5 schedule. Injection of 3.5-7.0 micrograms HP directly into the nucleus accumbens, or intra-accumbens injections of 6-hydroxy-dopamine, also decreased lever pressing for food and increased feeding on laboratory chow. Thus, interference with brain dopamine suppressed a highly active instrumental response for food, although the behavior of the animal was still directed towards food acquisition and consumption.

A reproducible method for the optimum infection of human mononuclear cells and lymphoid cell lines by herpes simplex virus type 1.

Parameters for the infection of human mononuclear cells (MNC) with herpes simplex virus type 1 (HSV-1) were investigated and a procedure was established which resulted in a reproducible optimum number of cells expressing virus-specific cell surface antigens. The number of cells infected was independent of sex of the donor and independent of whether the donor was HSV-seropositive or -seronegative. On the average 18 +/- 6% of HSV-infected MNC from any given donor expressed HSV-specific cell surface antigens. When the standard procedure was applied to a variety of lymphoid cell lines, a high percentage of cells of both B and non-T/non-B lines expressed HSV-specific cell surface antigens, whereas T-cell lines appeared resistant to HSV infection.

The anti-excitotoxic effects of certain anesthetics, analgesics and sedative-hypnotics.

Various agents were tested for their ability to antagonize the acute excitotoxic action of N-methyl-DL-aspartate (NMA) and kainic acid (KA) on neurons in the in vitro chick embryo retina. The following compounds (in order of descending potencies) were effective in completely blocking the neurotoxic activity of NMA: phencyclidine, ketamine, (+/-)-SKF 10,047, pentazocine, D-aminophosphonovalerate, D-amino-phosphonoheptanoate, D-alpha-aminoadipate, OH-quinoxaline carboxylate, kynurenate, (+/-)-cis-2,3-piperidine dicarboxylate, secobarbital, amobarbital and pentobarbital. The latter 6 agents also protected against KA toxicity but complete protection was observed only from relatively high concentrations. At 20 mM, Mg2+ blocked NMA toxicity but at concentrations up to 30 mM did not block KA toxicity. Compounds that failed to block either NMA or KA toxicity include D- and L-aminophosphonobutyrate, L-glutamic acid diethyl ester, xanthurenate, GABA and taurine. The chick embryo retina is a useful preparation for identifying agents that have either excitotoxic or anti-excitotoxic activity.

L-homocysteic acid: an endogenous excitotoxic ligand of the NMDA receptor.

L-Homocysteic acid (L-HCA) has been proposed as a natural transmitter at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor based on recent evidence that L-HCA occurs L-HCA occurs naturally in the mammalian CNS, is released from K+ stimulated brain slices in a calcium-dependent manner and may be contained in nerve terminals located in certain brain regions that have a high density of NMDA receptors. Here we report that L-HCA potently induces a pattern of cytopathology in the ex vivo chick retina which mimics the pattern of NMDA but not kainic acid (KA) neurotoxicity. We also show that known NMDA antagonists, including Mg++, D-aminophosphonopentanoate and certain anesthetics, analgesics, and sedative hypnotics block the neurotoxic actions of L-HCA in direct proportion to their efficacy in blocking NMDA neurotoxicity. While there is a perfect correspondence between agents that block NMDA and L-HCA neurotoxicity, only a few such agents are active against KA neurotoxicity. We find that 3H-Glu binding is inhibited more potently by L-HCA (Ki = 67 microM). Moreover the patterns with which L-HCA and NMDA displace 3H-Glu binding in autoradiograms appear essentially identical. These findings are consistent with the proposal that L-HCA is an endogenous ligand at NMDA receptors.

Ventrolateral striatal dopamine depletions impair feeding and food handling in rats.

The present study was conducted to characterize the changes in feeding behavior produced by localized depletion of dopamine (DA) in the nucleus accumbens and subregions of the neostriatum in the rat. Food-deprived rats were given at least 2 weeks of training, which consisted of being placed in a Plexiglas box and being given 15-18 g of food for a 30-min session. After the training period, rats received bilateral injections of the neurotoxic agent 6-hydroxydopamine (6-OHDA) into the nucleus accumbens, ventromedial striatum, or ventrolateral striatum. Observations were made in 30-min tests on days 3 and 7 after surgery, and measures were obtained for total food intake, time spent feeding, rate of feeding, and forepaw usage during feeding. The ventrolateral striatum was the only site at which dopamine depletion altered aspects of food intake. Rats with ventrolateral striatal DA depletion had reductions in food intake, decreases in the rate of feeding, and impaired forepaw usage during feeding. Time spent feeding was not significantly affected by DA depletion. Water consumption was significantly reduced by DA depletions in the ventrolateral striatum but not by depletions at other sites. These results indicate that ventrolateral striatal DA depletions decrease food intake by impairment of motor functions necessary for the performance of feeding behavior.

Calcaneal donor bone grafts.

Autogenous bone grafting is an important part of many foot and ankle surgical procedures. Although alternative bone graft materials such as allogeneic bone are available, autogenous bone continues to be the material of choice for many procedures. The calcaneus provides a source of small amounts of autogenous corticocancellous or cancellous bone. The author describes the surgical technique for procurement of calcaneal bone grafts. Twenty-five cases are reviewed. The morbidity associated with procuring calcaneal bone grafts is favorably compared with that associated with procuring bone from other donor sites.

Bone graft reconstruction of a flail digit.

The author presents a case report with a 1-year follow-up period demonstrating successful bone graft stabilization of an iatrogenic flail second toe. The author discusses the techniques for calcaneal autogenous bone grafting for reconstruction of the iatrogenic shortened toe combined with ancillary procedures to improve the digital length pattern. After 18 months, this staged approach to stabilization of the digit and realignment of the digital length pattern appears to be successful.

Recent concepts in understanding a child's pain.

The management of pain in children is a complex process that has significant differences from adult pain management. The authors describe the myth of children's lack of pain and how that myth has resulted in frequent undermedication of children's pain. The authors describe the physical and psychological aspects of pain. Assessment techniques for more accurately determining pain in children are also described. The authors conclude with a description of pharmacologic aspects of pediatric pain management.

Surgical repair of a talar body nonunion.

Fractures of the talus are significant injuries and are usually intra-articular. The authors discuss the evaluation and management of a patient with a delayed union of a talar body fracture. Assessment of talar vascularity and joint integrity should be performed preoperatively. The role of internal fixation and continuous passive motion is discussed.

Juvenile hallux valgus with compensated metatarsus adductus. Case report.

The authors describe a case report of a patient with compensated metatarsus adductus and juvenile hallux valgus bilaterally. Treatment included closing base wedge abductory osteotomies of metatarsals one through five, modified McBride bunionectomy, Evans calcaneal osteotomy, and a percutaneous tendo Achillis lengthening. The patient maintained excellent correction on the right foot after 6 years. Hallux valgus recurred on the left foot after 2 years. The authors attribute this recurrence to a significant internal femoral torsion on the left leg. The significance of superstructural deformities on juvenile hallux valgus is discussed.

Closed rupture of the anterior tibial tendon. A case report.

Closed subcutaneous rupture of the anterior tibial tendon is a relatively uncommon injury that requires a thorough clinical examination to diagnose correctly. The authors report a case of this disorder and provide a review of the relevant literature. A method of surgical repair not previously described in the literature is also presented.

Bone grafting in foot and ankle surgery. A review of 300 cases.

Three hundred foot and ankle bone grafts were reviewed in three separate series of 100 consecutive grafts from two institutions. The series represent a period from 1977 to 1990 and demonstrate treatment patterns that varied over time and between institutions in indications, graft material, and perioperative management. Over 42% of the 300 grafts were for calcaneal osteotomies; most were Evans calcaneal osteotomies. Over 72% of the grafts were allogeneic bone-bank bone, which performed well in calcaneal osteotomies and for packing of defects. Upon review of the incidence of bone complications, no significant differences were observed between surgical procedures that used autogenous versus allogeneic grafts. However, four out of six failures of first metatarsal repair were with allogeneic bone. There was a significant difference in complication rates for the major indications for bone-graft surgery. Nonunions and arthrodeses resulted in higher complication rates than expected, whereas calcaneal osteotomies resulted in a lower complication rate than expected.

Spinal morphine anesthesia and urinary retention.

Spinal anesthetic is a common form of surgical anesthetic used in foot and ankle surgery. Spinal morphine anesthetic is less common, but has the advantage of providing postoperative analgesia for 12 to 24 hr. A number of complications can occur with spinal anesthesia, including urinary retention that may be a source of severe and often prolonged discomfort and pain for the patient. Management of this problem may require repeated bladder catheterization, which may lead to urinary tract infections or impairment of urethrovesicular function. This study reviews the incidence of urinary retention in 80 patients (40 after general anesthesia and 40 after spinal anesthesia) who underwent foot and ankle surgery at Saint Joseph's Hospital, Philadelphia, PA. Twenty-five percent of the patients who had spinal anesthesia experienced urinary retention, while only 7 1/2% of the group who had general anesthesia had this complication. Predisposing factors, treatment regimen, and recommendations for the prevention and management of urinary retention are presented.

Hydroxyapatite as a bone substitute.

A bone substitute eliminates the need for autogenous and allogeneic bone grafting, along with the complications unique to each. Coralline hydroxyapatite is a synthetic bone void filler manufactured from marine coral, which has a natural trabecular structure similar to that of cancellous bone. While initial studies have been promising, the use of coralline hydroxyapatite may be limited in the foot and ankle owing to its inherent mechanical weakness and lack of biodegradation. In this retrospective study, 20 patients who received coralline hydroxyapatite implants were reviewed to determine associated complications and host reaction to the material. The results indicate that the material is biologically inert and safe to use as a small-defect filler in low-load applications. Long-term clinical trials are needed to determine the indications for this material in the foot and ankle.

Digital dissecting hematoma. A differential diagnosis.

The dissecting hematoma, a little mentioned postoperative complication following digital surgery, occurs when there is damage to the blood vessels intraoperatively. Accurate and immediate diagnosis is essential for providing early and appropriate treatment.

Characteristics of adolescent hallux abducto valgus. A retrospective review.

The authors review 63 surgical cases of adolescent hallux abducto valgus in 54 patients from the Foot and Ankle Institute of the Pennsylvania College of Podiatric Medicine over a 7-year period. Twenty-five closing base wedge and capital osteotomies were evaluated for radiographic changes. Three quarters of a group of 65 patients with adolescent hallux abducto valgus were found to have an abnormally high metatarsus adductus angle. Patients who had a closing base wedge osteotomy had higher preoperative radiographic angles for the intermetatarsal, metatarsus adductus, hallux abductus, and proximal articular set angles as well as the tibial sesamoid position. Those patients had 4.49 degrees more intermetatarsal angle correction than the patients who had a capital osteotomy. Implications for evaluation and treatment are discussed.