Omega 3 and omega 6 fatty acids in human and animal health: an African perspective.

Lipids are essential for plant and animal development, growth and nutrition and play critical roles in health and reproduction. The dramatic increase in the human population has put increasing pressure on human food sources, especially of those sources of food which contain adequate levels of polyunsaturated fatty acids (PUFAs) and more importantly, sources of food which have favorable ratios of the n-3 (18-carbon, α-linolenic acid, ALA) to n-6 (18-carbon linoleic acid, LA) PUFAs. Recent studies have demonstrated the beneficial effects of the n-3 PUFAs in diets as well as potentially negative effects of excessive levels of n-6 PUFAs in diets. This review discusses these human health issues relating to changes in diets based on environmental and industrial changes as well as strategies in East Africa for improving lipid composition of food using indigenous sources.

Endocrine control of mucosal immunity in the female reproductive tract: impact of environmental disruptors.

The complexity of the human female reproductive tract (FRT) with its multiple levels of hormonally controlled immune protection has only begun to be understood. Dissecting the functions and roles of the immune system in the FRT is complicated by the differential hormonal regulation of its distinct anatomical structures that vary throughout the menstrual cycle. Although many fundamental mechanisms of steroid regulation of reproductive tract immune function have been determined, the effects of exogenous synthetic steroids or endocrine disruptors on immune function and disease susceptibility in the FRT have yet to be evaluated in detail. There is increasing evidence that environmental or synthetic molecules can alter normal immune function. This review provides an overview of the innate and adaptive immune systems, the current status of immune function in the FRT and the potential risks of environmental or pharmacological molecules that may perturb this system.

Molecular approaches for the evaluation of immune responses to zona pellucida (ZP) and development of second-generation ZP vaccines.

It has long been established that there are major variations in both the immunogenicity and antigenicity of native zona pellucida (ZP) proteins. These differences appear to be more pronounced with respect to genetically engineered ZP proteins, which do not have native post-translational modifications (for example glycosylation and sulphation). As the number of animal species that are now included in population management programmes using native porcine zona pellucida (PZP) proteins expands, it is increasingly important to carry out studies to evaluate the immune response variations among different species as well as the individual variation within a species. In an attempt to compare these complex immune responses, we have evaluated antibodies from numerous species immunized with native, genetically engineered ZP and synthetic ZP peptides. Such an immunocontraceptive method could have great potential. These studies are critical not only for the development of predictable immune responses that result in permanent sterilization versus reversible contraceptive effects, but also for predicting which vaccinogens (native ZP protein versus genetically engineered ZP proteins) might have detrimental effects on animal and human populations.

Molecular analysis of a carbohydrate antigen involved in the structure and function of zona pellucida glycoproteins.

A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-beta-glycosidase, endoglycosidase F, or combinations of neuraminidase plus beta-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.

Effects of dietary flax seed and sunflower seed supplementation on normal canine serum polyunsaturated fatty acids and skin and hair coat condition scores.

This prospective study involved supplementing 18 normal dogs with flax seed (FLX) and sunflower seed (SUN) and evaluating their effects on skin and hair coat condition scores and serum polyunsaturated fatty acids (PUFA) concentrations. Skin and hair coat were evaluated in a double-blinded fashion using a numeric scoring system and serum PUFA concentrations were determined. Our hypothesis was that changes in serum PUFA concentrations are associated with improvements in skin and hair coat and that serum PUFA would provide an objective method for making dietary fatty acid supplement recommendations. Although a numerical improvement was found in hair coat quality in both groups, this improvement was not sustained beyond 28 days. The relative per cent of 18:3n-3 concentrations in serum phospholipids increased in the FLX treated dogs but these concentrations remained unchanged in the SUN treated dogs. Also, elevations in relative per cent of 18:2n-6 concentrations in serum phospholipids were seen in the FLX group. The ratio of serum polyunsaturated to saturated fatty acids also showed a transient increase. These increases preceded the peak skin condition score peak value by approximately 14 days. It was concluded that a 1-month supplementation with either flax seed or sunflower seed in dogs provides temporary improvement in skin and hair coat. These changes appeared to be associated with increased serum 18 carbon PUFA.

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.

The ovary as an immune target.

The ovary does not have a distinct morphologic barrier between the immune system and the developing gametes. This is in contrast to the testis in which the junctional complexes between the Sertoli cells form the blood-testis barrier. Whereas there are numerous factors, including genetic ones, associated with ovarian dysfunction, the immune factors have frequently been implicated in ovarian dysfunction. Much of our knowledge used to evaluate the immune system of the ovary has come from studies on the expression of the zona pellucida (ZP) proteins during ovarian development. Initial studies by Dunbar and colleagues demonstrated that immunization of rabbits with porcine ZP proteins (but not rabbit ZP proteins) would result in the generation of antibodies that inhibit sperm binding to the ZP and interfere with normal ovarian follicular development. In contrast to the rabbit and primate models, immunization of mice or rats with porcine ZP proteins does not have an effect on fertility or ovarian function although immunization of certain strains of mice with mouse ZP peptides and immune activator systems has been shown to result in ovarian pathology. Whereas immune inflammatory reactions have been observed in the mouse models, no such immune reactions have been observed in rabbit, guinea pig, or nonhuman primate models. Subsequent observations in nonhuman primates have shown that immunization of primates with ZP proteins expressed from cDNAs coding for the mouse and rabbit ZP2 (the mouse homologue has 60% amino acid identity with human ZP2) or the mouse ZP3 (the mouse protein has 67% amino acid identity with human ZP3) causes ovarian dysgenesis. In contrast, immunization of primates with recombinant rabbit ZP1 protein (the mouse homologue has 39% amino acid identity with human ZP1) does not affect nonhuman primate ovarian function or follicular development but will elicit antibodies that inhibit sperm binding to the primate ZP. These studies have collectively provided important information concerning the immunologic status of the ovary and demonstrate the species variations in immune responses to different ovarian immunogens.

Structure and function of the proteins of the mammalian Zona pellucida.

The zona pellucida (ZP) is the extracellular matrix that plays important roles in sperm-egg interaction. The ZP is composed of three major glycoproteins that exhibit heterogeneity due to extensive post-translational modifications including glycosylation and sulfation. Because of these modifications the nomenclature of ZP proteins from different species based on electrophoretic mobilities has been confusing. As the cDNAs and genes encoding the different ZP proteins have been isolated and sequenced, it is now possible to relate these ZP proteins according to gene families. Using the mouse ZP nomenclature, the ZP proteins from different mammalian species can be classified into three protein families: ZP1, ZP2, and ZP3. Although some of the structural domains of the ZP proteins of different species are conserved within each family, they exhibit distinct biological properties. In the mouse it has been established that ZP3 is the primary sperm receptor while ZP2 has secondary sperm receptor properties. In the pig, however, ZP1 has been shown to have sperm receptor activity similar to that observed in the rabbit and nonhuman primates. It is of interest that the human ZP2 and ZP3 gene families are 60-70% conserved with respect to the mouse ZP amino acid sequence, while the mouse ZP1 is only 39% conserved with respect to human ZP1. Such differences in protein structure and glysosylation may explain the marked species differences in the biochemical, physicochemical and immunochemical properties of the ZP. Studies have now shown that the proteins of the ZP are expressed in a stage specific manner and that there is increasing evidence that ZP proteins are expressed by both granulosa cells and the oocyte and may play a role in granulosa cell differentiation.

Human sperm-oocyte recognition and infertility.

The incidence of infertility related to both male and female factors continues to rise despite many advances in reproductive technologies. Some abnormalities in human gamete interaction have been shown to be due to defects in the sperm, and others have been attributed to defects in the zona pellucida (ZP). Our lack of understanding of molecular mechanisms involved in the interaction of human sperm with the ZP in fertile as compared with infertile females and males has been limited because of the unavailability of human oocytes and ethical restraints on experimental studies. It is becoming increasingly apparent that improved clinical assays are necessary for evaluating sperm-ZP interaction in order to assess the optimal procedures for successful fertilization and pregnancy. With advances in molecular biology, the genes encoding the three major human ZP proteins have been identified and complementary DNAs are available to begin to better evaluate the molecular basis of sperm-ZP interaction.

Mapping of dominant B-cell epitopes of a human zona pellucida protein (ZP1).

Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition. These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.

Primary structure of the helical domain of porcine collagen X.

The entire primary structure of the collagen X helical region is presented, including identification of the extensive post-ribosomal modifications by amino acid sequencing and mass spectrometry. As in collagen I, a single residue of 3-hydroxyproline was identified, but for collagen X this was located near the N-terminal end of the helix. Lysine residues in collagen X are extensively hydroxylated/glycosylated: at least 11 sites were localized and shown to be fully glycosylated, exclusively as glucosyl-galactosyl derivatives. The lysine-derived crosslinks, dihydroxylysinonorleucine and hydroxylysinonorleucine, were shown to be present in a 3:2 molar ratio primarily within the C-terminal portion of the helix.

The covalent attachment of polyamines to proteins in plant mitochondria.

Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases.

Guidelines for the pediatric perioperative anesthesia environment. American Academy of Pediatrics. Section on Anesthesiology.

The American Academy of Pediatrics proposes the following guidelines for the pediatric perioperative anesthesia environment. Essential components are identified that make the perioperative environment satisfactory for the anesthesia care of infants and children. Such an environment promotes the safety and wellbeing of infants and children by reducing the risk for adverse events.

It takes a village and time... a collaborative case study.

Today's approach to health care presents a challenge to those providers who work with patients with multifaceted complex issues that require the time and the efforts of several clinicians. These patients do not easily fit into a short-term, predetermined mold of care. This is an ongoing issue that warrants further investigation. This article presents a collaborative case study that chronicles the case of a patient who had multiple problems, such as homelessness, depression, substance abuse, anger, and serious emotional and physical scars contributing to a damaged self-image. In this case, three clinicians in three programs collaboratively assisted the patient in his complicated trek toward an improved quality of life.

From famine to feast: the role of methylglyoxal production in Escherichia coli.

The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen.

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.

Photoreceptor rim protein: partial sequences of cDNA show a high degree of similarity to ABC transporters.

PURPOSE: To isolate and sequence cDNA for bovine rim protein, a large membrane-bound glycoprotein found in photoreceptor outer segments. METHODS: Bovine rim protein was N-terminally sequenced (22 residues) and fragments were prepared by partial proteolysis. Two internal sequences of 21 and 18 amino acid residues were obtained from 35 kDa and 32 kDa fragments, respectively. Sense and anti-sense oligonucleotide primers were constructed, based on the peptide sequences derived from the 35 kDa and 32 kDa fragments, respectively, and the polymerase chain reaction (PCR) was used to amplify a 150 bp sequence from bovine retinal cDNA. RESULTS: The amplified sequence coded for the remainder of the peptide sequence determined from the 35 kDa fragment, which was not present in the primer, confirming that it was derived from the rim protein. The 150 bp sequence was translated to give a 50 amino acid peptide. Part of this peptide was compared with Dna sequence databases using the TFastA program, which found 94.6% identity with an EST derived from human retina and 86.1% identity to the mouse abc1 transporter. CONCLUSIONS: It is proposed that rim protein is a member of the ATP transporter family of proteins. It may be involved in transport of molecules involved in visual transduction across the photoreceptor disk membrane.

Identification of a meiotically expressed carbohydrate antigen in ovarian carcinoma: I. Immunohistochemical localization.

A monoclonal antibody developed against a meiotically expressed porcine oocyte carbohydrate antigen has been shown to recognize an antigen in ovarian surface epithelial cells. Immunohistochemical studies of ovaries demonstrated that this antigen is present in the ovarian surface epithelia (OSE) of numerous mammalian species, including the non-human primate and the human (1). Although most of the ovarian surface epithelial cells are lost during aging in the human, a few cells may remain in ovarian crypts. In view of theories that most ovarian carcinomas are derived from the OSE cells in aging women, the PS1 antibody has been used to evaluate ovarian tumors using immunocytochemistry to detect the PS1 antigen in paraffin embedded pathology tissues. The present study found that the PS1 antigen is abundant in a number of malignant ovarian tumors, but is not expressed in a non-malignant Brenner's (ovarian) tumor or granulosa cell tumors. This antibody therefore appears to have great potential for the histopathological and immunochemical analysis of ovarian tumors.

Identification of a meiotically expressed carbohydrate antigen in ovarian carcinoma: II. Association with proteins in tumors and peritoneal fluid.

A monoclonal antibody developed against a meiotically expressed porcine oocyte carbohydrate antigen has been shown to recognize an antigen in ovarian surface epithelial cells (OSE) of numerous mammalian species, including the non-human primate and the human (1). Although most of the ovarian surface epithelial cells are lost during aging in the human, a few cells may remain in ovarian crypts. Because the majority of ovarian carcinomas are thought to be derived from the OSE cells in aging women the PS1 antibody has been used to evaluate ovarian tumors. The secretory origin of this carbohydrate antigen in meiotic cells prompted further analyses of peritoneal fluid collected from gynecological surgery patients including those diagnosed with ovarian cancer. The present study demonstrates that ovarian tumor proteins separated on SDS PAGE include an antigen having a heterogeneous molecular weight (> 100 kDa) typical of glycosylated proteins. Additional studies show that peritoneal fluid from 19 patients not having cancer contain PS1 associated glycoproteins. However, of 14 cancer patients, only one had detectable levels of the carbohydrate antigen. These observations suggest that either the secretion of this glycoprotein is altered in ovarian carcinoma or that glycosidases or other proteolytic enzymes are involved in the degradation of these glycoproteins.