James Grant Thompson Memorial Lecture. Surgical management of poisonous snakebite.
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Biomedical subjects
Publications and source records attributed to T T Huang.
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Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.
The inner acrosomal membrane (IAM) develops during the spermatid stage of differentiation as that portion of the Golgi-derived acrosome granule that tightly associates with the condensing sperm nucleus. In some mammalian species, an electron-dense proteinaceous material accumulates between the IAM and the nuclear envelope, collectively comprising the "perforatorium." Evidence, including its partial purification and its structural resistance to detergents and sonication, suggests that the IAM is an unusually resiliant membrane. Dense paracrystalline arrays of intramembranous particles, a lack of lectin-mediated receptor modulation, and its lack of participation in sperm-egg fusion suggest that the IAM lacks the same degree of fluidity as the egg surface plasmalemma. Observations using monoclonal antibodies, however, suggest that some specific antigenic modulations may be possible within the IAM. Its structural rigidity is of obvious mechanical value during sperm penetration through the zone pellucida. An additional role as a scaffold for putative zona lysin material remains controversial. Biochemical evidence suggests that acrosin, for example, is not entirely soluble and that some remains sperm-associated, depending on the conditions of acrosome disruption. Nevertheless, morphological studies do not agree on acrosin's specific localization to the IAM. Currently there is only very limited information concerning the localization of the other acrosomal enzymes to the IAM. Another possible role for the IAM in some species may be in recognizing the zona pellucida. Evidence for this derives from the observation that fucoidin, a fucose heteropolysaccharide, inhibits guinea pig sperm-zona binding, and bound fucoidin can be localized to the IAM and equatorial regions of the living acrosome-reacted spermatozoa. Finally, the IAM may have a role in early recognition/adhesion with the colemma.
The purpose of this study was to examine how trypsin inhibitors affect the guinea pig sperm acrosome reaction in vitro. Using spermatozoa pretreated with lysophosphatidyl choline, we found that both naturally occurring high molecular weight and the smaller synthetic trypsin inhibitor p-aminobenzamidine (PAB) delayed the onset of the acrosome reaction as monitored by light microscopy. Examination with electron microscopy revealed that acrosomal matrix dispersal rather than membrane fusion was affected. Despite the morphologic delay in acrosomal content release, PAB unexpectedly permitted 96% of soluble acrosomal antigen to be released into the supernatant. In addition, total acrosin release in the presence of PAB was 74% of control, with the vast majority as latent rather than active enzyme. A morphologically intact but membrane-free target of acrosomal matrix (AM), which is sensitive to trypsin inhibitor, was partially purified using Triton-x-100 at pH 5.2. AM remained morphologically stable at pH 5.2; however, shift up to pH 7 resulted in rapid dissolution within several minutes as monitored by light and electron microscopy and light scattering. Trypsin inhibitor prevented dispersion of AM at pH 7. The results suggest that, during the acrosome reaction, one distinct region of the acrosomal contents disperses after membrane vesiculation in a pH and trypsin inhibitor-insensitive fashion while a pH sensitive trypsin-like activity (acrosin?) disperses another discrete region of acrosomal matrix.
To determine the changes in patterns of 17 beta-estradiol and progesterone levels underlying abnormal cycles in bitches immunized with solubilized crude porcine zonae pellucidae (cPZP), to attempt to circumvent these problems by immunizing with a purified zona fraction (pPZP), and to test the effectiveness of different adjuvants, bitches were immunized with cPZP or pPZP 2-6 times with no adjuvant, Freund's adjuvant, alum adjuvant, or the adjuvant CP-20,961. The bitch immunized without adjuvant had a low titer with a normal cycle and fertility. Immunization with cPZP and adjuvant produced moderate to high titers of antizona antibodies and infertility. Bitches with high titers experienced abnormal estrous cycles. Estradiol rose during proestrus, but instead of falling sharply in early estrus as in controls, it remained elevated. Progesterone did not rise. The moderate-titered bitches had normal cycles and steroid patterns. Bitches immunized with pPZP had moderate titers. Cycles were normal after 3 injections, but after 6 injections one bitch had an abnormal cycle. One pPZP-immunized bitch remained fertile but the others were infertile. Alum was the mildest adjuvant, causing no injection site lesions, but the highest titers occurred with Freund's and CP-20,961 adjuvants. All three adjuvants induced titers sufficient to inhibit fertility. Infertility in bitches immunized with PZP may be due to prevention of zona penetration, because their antisera inhibited zona penetration of oocytes by spermatozoa in vitro. However, alterations in ovarian function preventing ovulation and luteinization could be involved in high-titered bitches.
During the 5-year period between 1978 and 1983, a total of 56 individuals have undergone pharyngoesophageal reconstruction at our hospitals. To restore the continuity of the upper enteric tract, conventional skin flaps with or without the underlying muscle were used in 27 and a segmental free intestinal graft by means of microvascular technique was used in 29. Two-thirds of both groups received irradiation therapy before surgery. Fistula formation was encountered in 8 individuals who received a skin flap. This incidence was found to increase with the use of preoperative irradiation. In contrast, no patient developed a fistula among the 29 patients who received the segmental free intestinal graft.
The onlay cartilage grafting technique is described for treatment of unilateral or bilateral cleft lip nasal deformities. The alar cartilage is exposed through rim and intercartilagenous incisions. The cephalic half of the alar cartilage is excised, similar to the technique of traditional tip rhinoplasty. The harvested cartilage is applied to the intact caudal cartilage in layered fashion and secured with absorbable sutures. If necessary, successive layers may be added. These grafts provide a sturdy, yet delicate framework for a more normal appearing alar rim. We have performed this procedure on 16 patients, ages 10 to 41. Follow-up intervals range from 13 to 40 months, with a mean of 19 months. Results have been rated good-to-excellent by patients and surgeons. There has been no recurrence of the deformity. The only complication has been one nasal vestibule synechia.
Our previous study has shown that fucoidin, an algal heteropolysaccharide, is a potent inhibitor of sperm-zona binding in the guinea pig, hamster and human. To visualize the surface site of fucoidin binding, a biotinated derivative (B-Fuc) of the native fucoidin was prepared. B-Fuc retained the inhibitory activity and was used in conjunction with FITC-avidin to localize its binding sites on guinea pig spermatozoa using fluorescence microscopy. In living acrosome-reacted spermatozoa. B-Fuc bound predominantly to the inner acrosomal membrane and equatorial segment domains. The binding was effectively competed by a 10-fold excess of native fucoidin, but not by a 10-fold excess of heparin or a 20-fold excess of biotinated normal rabbit serum IgG. B-Fuc binding patterns on dead spermatozoa were quite different from that of living spermatozoa. The post-acrosomal region, rather than the inner acrosomal membrane and equatorial domains, was intensely labeled. This indicates the importance of using living cells in assessing true surface binding sites whenever possible. We conclude that the inner acrosomal membrane and/or equatorial domains are critical for zona binding in the guinea pig.
The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups. In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide (NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0. Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation; thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium. However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates of the SS/SH ratio.
In a study designed to evaluate the contraceptive potential of anti-egg zona pellucida immunization, bitches were injected with isolated and solubilized zonae pellucidae of either the pig or the dog in saline and Freund's adjuvant or with saline and adjuvant alone (controls). They were boosted monthly, and serum samples were collected before the first injection and 10 days after each injection. The titers of anti-zona pellucida antibodies in each serum sample were measured by treating fresh canine oocytes with the serum, then evaluating antibody binding as indicated by indirect immunofluorescence, precipitation of the zona surface, and penetrability of the zonae by spermatozoa in vitro. The bitches were bred when they came into estrus. All three bitches immunized with porcine zonae developed high titers (1:10,000 or more by indirect immunofluorescence) of antibodies that cross-reacted with canine zonae to cause precipitation of the zona surface both in vivo and in vitro and that completely inhibited penetration of the zonae by spermatozoa in vitro. The two bitches immunized with canine zonae developed only low titers, and their sera had little or no effect on treated zonae. The two control bitches did not develop anti-zona antibody. None of the bitches immunized against porcine zonae became pregnant when bred, but one bitch immunized against canine zonae and one control did become pregnant. The bitches immunized with porcine zonae had somewhat abnormal cycles for unknown reasons. Thus, we could not establish with certainty whether the infertility resulted from specific interference with fertilization, as in vitro, or from alterations in ovarian function, or both.
Murine intracisternal A-particles (IAP) are retroviruses expressed in large numbers in certain neoplastic cells and preimplantation mouse embryos. Immuno-electron microscopic detection of IAP-associated antigens in mouse rhabdomyosarcoma cells and early mouse embryos provides the first direct evidence that IAP produced by these two sources are serologically related. In both embryos and cells, data suggest that, after synthesis, IAP core protein rapidly associates with the endoplasmic reticulum. In addition, in embryos only, data confirm earlier observations that IAP-associated antigens are expressed on the cell surface. There is no evidence that the Golgi apparatus is involved in the processing of IAP antigens in either cell type.
Gynecomastia is a common deformity encountered among male patients. Surgery is primarily aimed at reducing the size of the breast mound and therefore should be considered aesthetic in nature. In an attempt to minimize the problems of hematoma formation, nipple necrosis, and unsightly scarring around the upper torso after the surgery, we have had the opportunity to utilize a circumareolar approach for breast tissue removal in 33 breasts (24 patients). This method, when compared with other techniques, has the advantage of leaving minimal scarring around the areolar margin while obliterating the dead space by imbricating the tissue stalk to minimize hematoma formation.
Immunoglobulin G and Fab antibodies were isolated from the serum of vasectomized guinea pigs, and the effects of the antibodies on fertilization in vitro were investigated. These antibodies had profound inhibitory effects on (i) sperm-to-sperm adhesion, (ii) the acrosome reaction, (iii) sperm-zona binding, and (iv) sperm-ovum fusion. This finding may explain certain cases of infertility after vasovasostomy in men.
We have examined the behavior of guinea pig spermatozoa towards the guinea pig zona pellucida in vitro. Experiments were designed to determine whether the state of capacitation or the acrosome reaction determined the ability of the spermatozoa to attach to the zona. Freshly collected cauda epididymal spermatozoa are acrosome-intact (AI) and, by definition, uncapacitated. These cells could not attach (at least persistently) to the zona. We then sampled spermatozoa from the progressively capacitating population at various time intervals. At no point did AI spermatozoa from such samples have the ability to attach to the zona. On the other hand, acrosome-reacted (AR) spermatozoa that appeared in increasing number during this capacitation period rapidly and firmly adhered to the zona. Adhesion by AR spermatozoa prepared in several ways culminated in successful zona penetration and fertilization of the ovum. The data provide unequivocal evidence that AR guinea pig spermatozoa attach to zonae and fertilize the ova. We find no evidence that AI spermatozoa firmly attach or bind to the zona, regardless of their state of capacitation.
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The dimension of the lower tarsus diminishes with aging. Changes in the number and size of the meibomian glands and in the character of intermingling fibroconnective tissues are thought to be responsible. Furthermore, such alterations of the structural integrity play an important role in the pathogenesis of entropion encountered commonly in the elderly. We have advocated the use of an autogenous cartilage graft harvested from the ear to correct the deformity, especially in instances where the conventional methods of correction have failed.
Murine intracisternal A-particles (IAP) are endogenous retrovirus particles expressed in large numbers in certain neoplastic tissues and during early stages of normal preimplantation mouse (outbred ICR) embryogenesis. IAP-associated antigens synthesized by preimplantation mouse embryos were precipitated by a rabbit (New Zealand White) antibody prepared against IAP from murine myeloma 104E. Characterization of the embryo immunoprecipitates with the use of one- and two- dimensional polyacrylamide gel electrophoresis revealed that a group of five proteins was synthesized by two- to eight-cell stages, but only three of these (molecular weight of 67,000, 69,000, and 73,000) correlated with the morphologic expression of IAP in embryos. ONly the proteins with molecular weights of 75,000 and 77,000 were detected in morulae and blastocysts, when embryos were IAP-negative. This is the first biochemical identification of endogenous retrovirus-associated antigens in preimplantation mouse embryos.