A role for Cdc42 in macrophage chemotaxis.

Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)-induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1-induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished. We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.

Murine epidermal growth factor (EGF) fragment (33-42) inhibits both EGF- and laminin-dependent endothelial cell motility and angiogenesis.

Laminin, murine epidermal growth factor (mEGF), and the synthetic laminin peptide Lam.B1(925-933) (a linear peptide from the B1 chain of murine laminin, CDPGY1GSR-amide) all stimulate endothelial cell motility above basal rates, whereas a synthetic mEGF fragment, mEGF33-42 (a linear peptide from the C-loop of mEGF, acetyl-C-[S-Acm]-VIGYSGDR-C-[S-Acm]-amide), inhibits motility. In both human SK HEP-1 and embryonic chick endothelial cells, mEGF33-42 blocks both EGF- and laminin-stimulated locomotion of endothelial cells. In vivo, mEGF33-42 also blocks both laminin- and mEGF-induced angiogenesis in the chick. In the human cell line. Lam.B1(925-933) has an additive effect in coincubation with either laminin or mEGF, but it blocks their effects in the chick cells. Lam.B1(925-933) alone stimulates angiogenesis in the chick but blocks laminin-induced angiogenesis. Thus, mEGF33-42 acts as a general laminin antagonist, whereas Lam.B1(925-933) acts as a laminin agonist in human cells, but in chick cells it acts as a partial antagonist. We propose that the presence of an anionic group at the eighth residue of mEGF33-42 may be the source of the antagonistic effects seen with this peptide as compared with the laminin fragment. These findings have important implications in the design of human antiangiogenic agents, and also in the use of chick models in the study of human disease.

Rho family proteins and cell migration.

The GTP-binding proteins Rho, Rac and Cdc42 are known to regulate actin organization: Rho induces the assembly of contractile actin-based filaments such as stress fibres, Rac regulates the formation of lamellipodia and membrane ruffles, while Cdc42 is required for filopodium extension. All three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. Cell migration involves co-ordinated and dynamic changes in the actin cytoskeleton and cell adhesion, and we have therefore investigated the roles of Rho family proteins in migration, using two model cell systems. First, in the macrophage cell line Bac1, Rho and Rac were found to be required for colony-stimulating factor-1 (CSF-1)-induced cell migration. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient. This implies that Cdc42, and probably filopodia, are required for gradient sensing and cell polarization. Secondly, in the Madin-Darby canine kidney (MDCK) epithelial cell line, Rho and Rac are also essential for migration induced by hepatocyte growth factor/scatter factor. Rac is required for lamellipodium formation and is apparently activated via Ras. Interestingly, however, Rac does not induce lamellipodium formation in unstimulated MDCK cells, indicating that Rac signals differently in epithelial cells compared with fibroblasts or macrophages. Our results point to central roles for Rho, Rac and Cdc42 in co-ordinating cell migration.

Gray scale ultrasonography, computerized tomography, and nephrotomography in evaluation of polycystic kidney and liver disease.

A comparison of gray scale ultrasonography and computerized axial tomography in adults with known adult-type polycystic disease and of ultrasonography and high-dose nephrotomography in their progeny is being conducted. Although all three modalities have proved capable of demonstrating cysts of the kidney and liver, ultrasound has been the most consistent in identifying these lesions. Ultrasound is valuable in diagnosing polycystic disease in adult with large, poorly functioning kidneys; in addition, since cysts could be identified by ultrasound in children who had normal nephrotograms, it provides a safe and useful method of obtaining information for genetic counseling.

Orbital hemorrhage with loss of vision.

A 58-year-old woman had the sudden onset of unilateral painful proptosis, ophthalmoplegia, vomiting, and loss of vision. Computed axial tomography showed a mass that was greatly attenuated in the orbit. The initial reading of the internal carotid angiogram was normal, but a subtraction study showed a hypervascular lesion within the orbit with features indicating a hemangioma. Orbital decompression failed to restore the vision as intraorbital hemorrhage had irreparably damaged the optic nerve.

Preoperative laminagraphic evaluation of the sphenoid sinus for transsphenoidal surgery.

In recent years the transethmoidal and transnasal approaches to the pituitary gland have become increasingly popular. In each case, detailed preoperative analysis of the anatomy of the sphenoid sinus and its boundaries is crucial in facilitating entry to the pituitary fossa and in reducing intraoperative complications. Certain operative difficulties and complications are the result of the marked variability in the anatomy of the sphenoid bone and its sinus. These include: loss of orientation within a capacious sinus; perforation of the carotid artery or cavernous sinus; injury to the optic nerve or ophthalmic artery; and violation of the subarachnoid space with resultant pneumocephalus or cerebrospinal fluid rhinorrhea. Pneumatization develops originally within the presphenoid, beginning anteriorly and proceeding posteriorly. Around the time of puberty the pneumatization usually penetrates up to the spheno-occipital synchondrosis. Dense bony septa remain within the sinus along the plane of fusion of the various sphenoidal segments. Preoperative laminagraphy of these ridges and septa is extremely helpful in planning the safest and most direct route to the sella. Laminagraphy is preferable to routine skull films because the septal detail is unobscured by overlying facial structures. Pleuridirectional laminagraphic motion (circular or hypocycloidal) is preferred to linear motion because of the clarity and detail that they provide.

Effect of mannitol on the traumatized spinal cord. Microangiography, blood flow patterns, and electrophysiology.

The effects of mannitol on the spinal cord blood flow patterns in experimental traumatic paraplegia were correlated with microangiographic and electrophysiologic studies. At 1 hour following a therapeutic dose of mannitol (3 g/kg), an improved fluorescent intramedullary vascular pattern was detected among the mannitol-treated animals relative to those that were not treated. Within 4 hours, perfusion of many areas of the lateral white matter of the spinal cord often approximated normal in the mannitol-treated group. This pattern of perfusion was in striking contrast to that seen in the spinal cord of untreated animals, which displayed an almost total lack of fluorescing vessels at this later time. These findings correlated with an increased vascular caliber as revealed by microangiography and were postulated to be the result of a decrease in vasospasm and an expanded intramedullary blood volume following the administration of mannitol. Although mannitol therapy did not reverse the loss of the cortical evoked response observed during the 4-hour interval studied, the observation of improved blood flow patterns in the white matter is encouraging and warrants further study.

Duration of symptoms and disc configuration in chemonucleolysis.

Previous studies have compared the effectiveness of chemonucleolysis with surgery, but currently, no objective criteria have been correlated with the clinical outcome. The authors reviewed 28 cases where the patients had undergone chymopapain injection to determine the significance of disc herniation size, disc space height reduction, and the duration of symptoms on clinical outcome. All patients had a complete history, physical examination, and discogram, and most had pre- and post-injection computerized tomography (CT) or myelogram. Nine of the 28 patients were considered clinical failures. Seven underwent laminectomy and discectomy and were improved markedly. Two patients were advised to have surgery but refused and were considered clinical failures. The causes of failure were unknown in three patients, free fragment in two patients, and diabetic neuropathy in one. Only two patients who did well showed complete resolution of the disc deformity on repeat CT scan. The remainder still had evidence of an avascular deformity that persisted although reduced in size. The failures showed no changes in disc size. The height of the disc space was too variable to be correlated with clinical outcome. Patients who failed had a longer duration of symptoms than the ones who did well (15.1 months for failures vs. 5 months). Therefore, some reduction of disc deformity size, but not necessarily complete reduction, is necessary for a good result, and the enzyme is not as effective in patients with long-standing symptoms.

Small hemorrhages vs. small calcifications in brain tumors: difficulty in differentiation by computed tomography.

The difficulty in differentiating multiple small hemorrhages from multiple small calcifications in brain tumors by computed tomography (CT) is discussed. Illustrative CT scans are presented and the theoretical basis for this problem is elaborated. The difficulty is attributed to the relatively large size of the voxel and the ensuing partial volume effect, particularly with small intraparenchymal lesions of different sizes and attenuations.

The effect of weight on intravaginal pressure and vulval conformation of mares.

Pressures were recorded monthly at two sites in the vaginas of nine horse mares with normal vulval conformation. The pressures were lowest when the mares' weights were minimal, and there was evidence of a descending pressure gradient from the vestibule to the cervix. Loss of body weight was also associated with shortening and angulation of the vulva, although it was not displaced above the pelvic brim.

The effect of pregnancy and foaling on intravaginal pressure in pony mares.

Pressures were recorded monthly at two sites in the vagina of each of five pregnant and five nonpregnant pony mares; pressures in five mares were also recorded weekly after foaling. The developing pregnancy did not influence pressure, and after foaling the integrity of the seal formed by the caudal reproductive tract was rapidly restored.

Genital tract pressures in mares II. Changes induced by oxytocin and prostaglandin F(2)alpha.

The effects of oxytocin, prostaglandin F(2)alpha and a prostaglandin F(2)alpha analogue on uterine and vaginal pressures in the mare were measured using electronic catheter-tipped pressure transducers. Catheterisation for 70 minutes produced no significant change with time. Oxytocin caused a rapid rise in intrauterine pressure which had subsided 20 minutes later. Cloprostenol (prostaglandin F(2)alpha analogue) caused an increase in uterine pressure which started ten minutes after administration and lasted for the duration of the recording (60 minutes post-injection). Prostaglandin F(2)alpha produced a uterine pressure increase ten minutes after administration which declined over the next 40 minutes. The activity of the three drugs was not consistently affected by reproductive status (oestrus, dioestrus or anoestrus). There were no significant drug effects on intravaginal pressure.

The use of catheter-tipped pressure transducers for chronic measurement of genital tract pressures in the ewe. II. Effects of oxytocin and prostaglandin F(2)alpha.

Chronic catheterisation of the uterus, ampulla, and abdomen was performed in five ewes using solid-state, catheter-tipped pressure transducers. The catheters remained in place for up to 129 d, allowing in vivo studies of the effects of oxytocin and prostaglandin F(2)alpha (PGF(2)alpha). These agents did not produce any measurable increase in abdominal pressure. Intravenous (i.v.) oxytocin elicited a rapid increase in work done by both the uterus and ampulla. Intramuscular (i.m.) PGF(2)alpha produced a delayed uterine response but little change in the ampulla; i.v. PGF(2)alpha produced a rapid response at both sites. Low plasma progesterone concentrations (< 0.5 ng/ml) were associated with a greater uterine and ampullary response to oxytocin and with an enhanced uterine response to PGF(2)alpha. However, the uterine tube response to intravenous PGF(2)alpha was greatest when plasma progesterone concentrations were high.

Genital tract pressures in mares. I. Normal pressures and the effect of physiological events.

A computerised system involving electronic catheter-tipped transducers was used to measure pressures developed within the vagina and uterus of mares. Larger mares tended to have lower intravaginal pressure than ponies. Insertion of an arm into the vagina to place the catheters caused a rise in intravaginal pressure. Pressure in the uterus was usually higher than that in the vagina. Both vaginal and uterine pressures were increased by urination, snorting, whinnying, stretching and respiration - the latter effect was most noticeable in larger mares. Vaginal pressure was also influenced by peristalsis in the rectum, defaecation, the passage of flatus and the stance of the mare.

Simplified irradiation dosimetry in carcinoma of the cervix (external irradiation and one radium insertion).

Gynecologic radiation oncologists have long desired a single unit by which radiation doses to specific reference points in the pelvis can be summed. The Patterns of Care Studies, Marcial's 1976 report to the Conference on Radiation Oncology of the American Cancer Society, and other radiation oncologists advise summed doses to Point A, Point B, or the pelvic side wall. Since there is a difference in the biologic effect of high intensity, long time interval teletherapy and low intensity, short time interval brachytherapy, the rad dose obtained from each modality cannot be simply added. Ellis and Sorenson in the Nominal Standard Dose (NSD) concept have described radium conversion factors that can be used to normalize brachytherapy rads at selected reference points to equivalent teletherapy rad. The two rad doses can then be summed. A simple method found useful in 50 cases of carcinoma of the cervix, and used with a computer or readily available reference tables and with any radium system, is described and discussed. Further clinical uses of the method are suggested.

Effect of repeated biopsy sampling on endometrial concentrations of sodium benzylpenicillin following intramuscular injection in pony mares.

Endometrial concentrations of sodium benzylpenicillin were determined in biopsy samples from anoestrous pony mares after intramuscular administration of the penicillin at 22,000 iu/kg. Serum samples, collected at the same time as biopsies, were also assayed for their penicillin content. In experiment 1 a single pair of blood and tissue samples was taken from each mare between five and 360 minutes after injection. In experiment 2 each mare was sampled consecutively six times (between five and 300 minutes) after injection. Both serum and endometrial concentration reached a peak between 15 and 30 minutes after injection. Tissue concentrations were lower than serum, except after 180 minutes in experiment 2. This suggests that at the time intervals used in the present study, serial biopsies eventually caused an increase in the endometrial content of penicillin.

Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient.

A rapid single-step procedure for isolation of equine neutrophils (PMNS) from peripheral blood is described. A discontinuous gradient of two Percoll solutions (densities of 1.087 kg litre-1 and 1.108 kg litre-1 was used. The PMNS were isolated to more than 95 per cent purity with a viability of more than 99 per cent and a cell recovery of more than 83 per cent. The method used was rapid and reproducible and the equipment required is relatively simple. The function of the recovered cells was assessed in a chemotactic assay using a modified Boyden chamber technique.

An investigation into the origin of the first fraction of the canine ejaculate.

Histological examination of the reproductive tract of the male dog failed to identify glandular tissue other than the prostate gland and its disseminated portion. A biochemical study of the three fractions of the ejaculate demonstrates similarities between the first and the third fractions. The composition of three ejaculates collected over 12 hours indicated that storage of prostatic secretion was a possible explanation for the biochemical differences observed. It is suggested that both the first and third fractions originate from the prostate gland.

Plasma concentrations of sodium benzylpenicillin after intrauterine infusion in pony mares.

Plasma concentrations of sodium benzylpenicillin were measured following intrauterine infusion at a dose rate of 22,000 u/kg (250,000 u/ml). The reproductive status of the mare at the time of infusion did not appear to influence plasma concentrations of penicillin, but preswabbing the endometrium for bacteriological culture resulted in peak plasma concentrations which were nearly twice those found in unswabbed mares.