Morphological aspects of the hypothalamic-hypophyseal system

Summary Enzyme-histochemical methods were used to study the metabolic activity of specialized ependyma of the ventrolateral walls and floor of the third ventricle in young male and female rats during the critical period of sexual differentiation of the hypothalamus (one week after birth). Histochemical tests were conducted for glutamic dehydrogenase, lactic dehydrogenase, glucose-6-phosphate dehydrogenase, glycerophosphate dehydrogenase and NADH₂-dehydrogenase. Enzyme activity was judged by cytospectrophotometry. All the data were treated statistically.It was found that the specialized ependyma of the ventrolateral wall and floor of the third ventricle (median eminence) in rats differed in their enzyme behaviour in males and females during the critical period of sexual differentiation of the hypothalamus. At the level of the arcuate nucleus ( ²-tanycytes) and the medial part of the median eminence ( ²-tanycytes) the ependyma was characterized by similar indices of metabolic activity in males and females in the decisive terms of the critical period (days 3, 5, and 7). On day 5 metabolic activity of these cells was reduced both in the males and in the females. Prominent sexual differences in the intensity of the enzyme reactions studied were noted in the ependyma of the lateral parts of the median eminence ( ¹-tanycytes) in the critical period. On day 5 metabolic activity of ¹-tanycytes was reduced in males and increased in females. It is suggested that these differences are caused by the receptor nature of ¹-tanycytes and suggest their implication in the mechanisms of sexual differentiation of the hypothalamus.     

Location of neurons projecting to the hypophysial stalk — median eminence in ring doves (Streptopelia roseogrisea)

The median eminence/pituitary stalk represents the final common pathway for fibers from neurons that project to the pituitary gland. We have used the lipophilic fluorescent tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) to determine the location of neurons projecting to the median eminence/pituitary stalk in ring doves. The tracer can be precisely applied to fixed tissue, in areas to which it is otherwise difficult to gain access. Follwwing application of DiI to the median eminence/pituitary stalk, labeled neurons were detected in six distinct regions: the ventromedial hypothalamic nucleus, paraventricular nucleus, supraoptic nucleus, in and ventral to the lateral forebrain bundle, preoptic area, and lateral septum. Labeled fibers branched extensively in the diencephalon, particularly along the third ventricle and in the septal-preoptic area. Sparse fiber labeling occurred caudal to the tuberal hypothalamus, even though these regions were close to the application site of the tracer. Labeled cerebrospinal-fluid-contacting cells were seen in the paraventricular region of the third ventricle. The results indicate that the avian neuronal system that projects to the median eminence and neural lobe occurs in diffuse clusters largely along the midline region of the hypothalamic septal-preoptic area. The paucity of fiber staining caudal to the tuberal hypothalamic region indicates that cells of these regions do not project to the median eminence/pituitary.     

Fetal hypothalamic transplants in the third ventricle of the adult rat brain

Summary Blocks of anterior hypothalamus were transplanted from 19 day-old fetuses of Wistar/Lewis rats into the third ventricle of adult male Brattleboro rats. Physiological changes in graft recipients and in sham-operated animals were monitored daily. Twenty days after surgery, the graft recipients and shamoperated animals were killed and their brains examined by correlative scanning and transmission electron microscopy. Host animals that exhibited both decreased polydipsia and increased urine concentration were found to have viable grafts within the third ventricle. The observed physiological changes suggested that synthesis and release of vasopressin occurred in the transplanted neurons. Grafts were well vascularized by vessels arising from the host hypothalamus. Neurons, with perikarya ranging from 8 to 30 m in diameter, glial cells, and neurites were located throughout the transplants. A neurohemal contact zone, similar to that normally seen in the median eminence, could not be demonstrated in the grafts. The absence of complete glial and ependymal barriers indicates a relatively close association between cells in the transplants and the cerebrospinal fluid. A large increase in supraependymal neurons and their processes, including an eruption of neurons through the floor of the third ventricle in one animal, was observed in graft recipients but not in shamoperated animals.Supported by NIH Grants NS 15109 and NS 13717     

Changes in the LHRH-immunoreactivity of hypothalamic structures during late pregnancy and after parturition in the guinea pig

Summary LHRH-immunohistochemistry and radioimmunoassay of the gonadotropins were used to demonstrate changes in the LHRH content of the hypothalamus during late pregnancy and after parturition in the guinea pig. Immediately after parturition a decrease in the amount of LHRH-associated fluorescence was observed in the medial preoptic area. Only 12 h later similar changes were found in the median eminence. In the precommissural region no obvious changes were noted in the quantity of LHRH-immunoreactive terminals and synapses. The radioimmunoassay of the gonadotropins shows an increase of LH shortly after parturition which reaches the level of a preovulatory rise, whereas the amount of FSH does not vary significantly. The role of the medial preoptic area and the OVLT in the regulation of the estrous cycle through LHRH is discussed.This work was performed under a postdoctoral fellowship at the INSERM unité 156, Lab. d'Histologie, Faculté de Médecine, Lille, France     

Tanycyte and vascular patterns in the basal hypothalamus of Coturnix quail with reference to their possible neuroendocrine significance

Summary A well developed system of ependymal glial cells with long basilar processes stretching to the surface of the brain (tanycytes, Horstmann, 1954) has been described in the basal hypothalamus of Coturnix quail. The tanycytes both in the median eminence and the ventro-lateral hypothalamus form a link between the third ventricle and the hypophysial circulation. The processes of the ventro-lateral tanycytes terminate in the region of the infundibular sulcus in apposition to a loose network of vessels which are continuous with the primary plexus of the hypophysial portal system.Within the median eminence, the subependymal capillary network connects the vasculature of the contra-lateral sides of hypothalamus. There are no direct connections with the hypophysial portal vessels.With the aid of the light and electron microscope the ventricular ependyma was divided into a dorsal typical region and two glandular regions (ventro-lateral and ventral). Each region contains different forms of tanycyte. One of the two forms of tanycyte (designated type 3) associated with the ventro-lateral glandular ependyma has no contact with the third ventricle.Ultrastructural studies on the glandular ependyma failed to show any obvious differences between castrated, oestrogen or testosterone implanted, and sexually mature or immature quail.The possibility that the tanycyte-vascular system may have a neuroendocrine role is discussed.I am indebted to Professor A. Oksche, Dept. of Anatomy, University of Giessen for providing research facilities and to The Royal Society for additional financial support.     

Localization of luteinizing hormone β-mRNA by in situ hybridization in the sheep pars tuberalis

Summary The localization of luteinizing hormone beta (LH)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded ³²P-or ³⁵S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by using a specific LH-antiserum indicated that the labelling of LH-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH subunit, but that the level of translation greatly varies according to the location of the cells.     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Evidence that lipolytic peptide B occurs in the ACTH/MSH-cells of the pituitary and in the brain

Summary Several lipid-mobilizing peptides occur in the pituitary, among them -lipotropin and lipolytic peptide A and peptide B. The latter two peptides are distinct from -lipotropin and appear to be chemically related to the neurophysins. Immunohistochemistry has now revealed that the lipolytic peptide B of the pituitary is localized in the ACTH- and MSH-cells. In addition, immunoreactive peptide B was found in axons of the posterior lobe of the pituitary. Immunoreactive peptide B was found also in nerve fibers and nerve cell bodies in the hypothalamus, particularly in the hypothalamo-hypophyseal tract and in the magnocellular neuronal system. Immunoreactive nerve fibers were numerous also in the periventricular nucleus of the thalamus. The antiserum against peptide B cross-reacts with neurophysin I, and hence, it cannot be excluded that at least part of the immunostaining in the brain reflects the presence of the latter component. However, the regional distribution of immunoreactive peptide B and neurophysin was not identical. Therefore, it is possible that authentic peptide B occurs not only in the pituitary but also in the brain.     

A scanning electron-microscopic study of “supramarginal cells” in the pituitary cleft of the rat

Summary Unusual cells lying above the marginal cellular layer of the rat pituitary cleft were studied by SEM and TEM. These cells — from their location termed supramarginal cells — have a characteristically irregular cell body from which arise a number of long and thin branched processes ending among the microvilli and cilia of the marginal cells delimiting the anterior and posterior walls of the cleft. Some supramarginal cells are star-shaped elements with thin extensions, others have a triangular or spindle-shaped body from which emerge long ameboid processes with fibril-like projections. Miniblebs, miniruffles, occasional veils and short microvilli extend over the surface of these elements.Supramarginal cells are very similar to the Kolmer epiplexus cells originally found on the choroid plexus or other areas of the third ventricular wall where they are known as supraependymal cells.Present evidence suggests that supramarginal cells of pituitary cleft might have phagocytic properties and an hematogenous origin as monocytes and, as such, closely resemble Kolmer epiplexus cells of brain ventricles. Others might arise from folliculo-stellate cells or closely related marginal cells once they become free into the pituitary cleft.Supramarginal cells are recognized as motile phagocytes acting as scavengers and possibly regulating the extracellular environment of the cleft and associated adenohypophy-sial tissues. In considering the area where they are located supramarginal cells might assume a specific role in relation to the integrated neuroendocrine function of the hypothalamus-ventricle-hypophysial complex. To this complex the pituitary cleft is closely associated embryologically and anatomically.     

Substance P-related peptides in the hypothalamus of Amphibia

Summary The occurrence of Substance P-(SP)-related peptides in the hypothalamus of three species of Amphibia (newt, clawed, toad, frog) was studied immunohistochemically employing the indirect immunofluorescence method or a double-step technique (indirect immunofluorescence followed by the peroxidase-antiperoxidase complex method). SP-like immunopositive fibers are seen throughout the hypothalamus. They are especially abundant in the preoptic area and in the outer zone of the median eminence, suggesting a role of SP-related peptides in the hypothalamo-hypophysial regulation in these animals. Some SP-like neurons are seen in the posterior hypothalamus and in the preoptic area. In the newt, such SP-like immunopositive neurons occur frequently in the preoptic periventricular grey.Work performed under the C.N.R. project Biologia della Riproduzione     

Effects of acute opiate-peptide administration on pro-opiomelanocortin cells of the intermediate lobe of the rat pituitary

Summary The effects of acute injections of synthetic opiate peptides into the lateral cerebral ventricle of young adult male rats on cells of the intermediate lobe of the pituitary were studied. Met-enkephalin (100/g) injected into anesthetized rats, or 20 g beta-endorphin administered via a previously implanted cannula to unanesthetized animals, will lead to cell degranulation and often to expanded Golgi zones and prominent regions of rough endoplasmic reticulum in secretory cells when tissue is fixed 45–60 min after peptide administration. Treatment of animals with the opiate antagonist naloxone hydrochloride prior to enkephalin injection appeared to prevent the cellular changes elicited with peptide alone. Observations suggest that opiate peptides administered to the cerebrospinal fluid may stimulate release of pro-opiomelanocortin-peptide from pituitary cells.     

Ultrastructural study of neurovascular contacts in the median eminence of the rat

Summary In the median eminence of the rat axons of the supraoptic-paraventricular-hypophyseal tract with elementary neurosecretory granules (150–200 m) traverse the internal zone. Terminals containing dense core vesicles 60–120 m in diameter end on the portal capillaries of the median eminence. A unique organisation of the primary portal capillaries is shown. Endothelial cells have many fenestrae. The pericapillary space has numerous extensions all of which represent a special zone around the endothelial tube. The fine structural organisation and function of the neurovascular contacts in the median eminence are discussed.     

Lectin cytochemistry of cell types in human and canine pituitary

Summary Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for -l-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal -galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal -galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal -galactose in corticotrophs, presence of terminal -galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal -galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.This research was supported by NIH Grants AM-10956 and HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant #79     

Schliffe von vogel-eischalen im dunkelfeld

Zusammenfassung Die taxionomisch geregelte Schichtung der Kalkschale des Vogeleies wird wesentlich durch Anwesenheit und Menge der Einschlusse bestimmt, sei es, daß Globularinklusionen allein auftreten oder neben ihnen Spaltinklusionen. Diese Umstände lassen sich am besten im Dunkelfeld an Dünnschliffen ermitteln, wobei Auskochen der Schliffe in Kalilauge die Spaltinklusionen optisch zum Verschwinden bringt. Das Verhalten der Einschlüsse wird an einigen Beispielen vorgeführt. Weiter erleichtert das Dunkelfeld, das Muster des Schalenkalkes an der Anordnung der Inklusionen zu erkennen, was an Quer- und Flachschliffen der StrauBen Eischale für Kegel- und Säulenlage erläutert wird. Vergleich der Befunde im Dunkelfeld mit jenen beim Phasenkontrastverfahren und im Polarisationsmikroskop lassen das Wesen der sog. trüben Dreiecke in den Kegeln der Straußen-Eischale klar erkennen.

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Summary The taxionomically ordered stratification of the avian egg shell is essentially determined by presence and quantity of the inclusions, may be that only appear globular inclusions or besides them cleft inclusions. These circumstances are ascertainable the best on thin ground sections in dark field whereby boiling of the sections in potash-solution let dis appear optically the cleft inclusions. Some examples illustrate the situations of the inclusions. Farther the dark field lightens to perceive the pattern of the shell calcite, indicated by the arrangement of the inclusions; this will be explained on transverse and tangential ground sections of the Struthio egg shell for the cone- and the column-layer. Comparison of the statements in dark field with them in phase contrast and polarization microscope show clearly the nature of the troubled triangles in the cones of the Struthio egg shell.

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Herrn Professor Dr. W. E. Ankel zum 70. Geburtstage.     

The rotifer trap of Zoophagus

The predacious watermold Zoophagus insidians traps loricate rotifers on short, lateral branches of the main, hyphal axis. These branches or traps are packed at their distal ends with a number of vesicles filled with an electron-dense matrix. Electron micrographs of the mycelium disclose a two-layered wall; the outer layer is electron dense and the inner, electron transparent. The outer dense layer on the tip of the trap is organized into a number of fine ridges and occasional discontinuities. Thin sections through recently trapped rotifers indicate that the cilia of the animals are stuck to the trap by a glue. This adhesive is derived from secretion of the matrix of the vesicles aggregated in the tip of the trap. The secretion mechanism is triggered by the animal and is accompanied by: 1. The separation of the two layers of the wall, 2. fusion of the vesicles with the cell membrane and 3. extrusion of the glue through pits in the tip of the inner wall of the trap. After snaring a rotifer, the previously arrested branch grows as a haustorium into the body cavity of the animal. The host tissues disintegrate within a few hours and appear to be the main nutrient source of the fungus.     

Cellular and subcellular 3H-estradiol localization in the pituitary by autoradiography

Summary The cellular and subcellular localization of 6.7-³H-estradiol-17 in the pituitaries of five immature and two mature castrated female rats was studied by autoradiography. The autoradiographic technique used minimizes or eliminates translocation. It is based on low temperature tissue preparation of freeze-dried sections which are dry-mounted on dried photographic emulsion, excluding known sources of translocation artifacts such as liquid fixatives, dehydrating and clearing fluids, embedding media and thawing. The animals were given from 0.093 to 0.63 g of ³H-estradiol and were sacrificed at 15 min, 1, 2, or 6 hours after the injection. A large portion of the anterior pituitary cells was found to be labeled; the extent of this labeling varied with dose, time of sacrifice after the injection, and photographic exposure time, but apparently not with the endocrine status of the animal. The portions labeled were 76 and 86 per cent for the immature and mature rats respectively, exceeding single tinctorial light-microscopic groups. Gomori trichrome chromophiles and chromophobes, cells with intense and weak pyronin basophilia, as well as morphologically defined castration cells, were all partially labeled and unlabeled. Acidophiles appeared to be labeled in a somewhat higher proportion. Cells of the intermediate and posterior lobe were generally unlabeled except for occasionally interspersed cell groups or single cells, especially at the border between intermediate and posterior lobe, probably identical with basophilic invaginations in man and other mammals. The subcellular concentration of radioactivity was always nuclear. The findings are interpreted as suggesting a) feedback control on the pituitary level, in addition to the diencephalic level, b) pluripotentiality of anterior pituitary cells, and c) possible positive feedback mechanism of estradiol with secondary negative effect. Dry-mount autoradiography with labeled hormones, as applied in this study, provides a new methodological approach to the elucidation of pituitary physiology and pharmacology.Supported by USPHS Grants 1-ROl-AM-12, 649-01, GRS Grant FR-5367, ACS Grant IN-41-H, and Otho Sprague Memorial Institutional Grant. — The author thanks Dr. N. S. HALMI for consultation.     

Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats

Summary Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immungold techniques also localized intracellular laminin within biosynthetic organelles and light body vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin ( 15 ng/gland) in gonadectomized rats than in controls ( 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ ( 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was spliced into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Muscle fibre differentiation and vascularisation in the juvenile European eel (Anguilla anguilla L.)

Summary The development of the pituitary gland and its blood vessels is described in rat embryos (gestational day 12 through day 21) injected with India-ink via the umbilical vessels. The vascularization of all components of the pituitary gland develops from the surface network covering the prosencephalic vesicle. However, vascular connections exist between the prospective median eminence and the anterior pituitary gland in the earliest stages examined (day 12) but are not augmented by vessels from the stomatodeal roof until day 13. Primary portal veins appear initially on day 13, the vascularization of the pars distalis is visible on day 15. The Mantelplexus covering the floor of the diencephalon is discernible on day 16. Large-caliber portal veins appear immediately before birth, but otherwise there is no significant change in the vascular pattern during the last five days of gestation. The pars intermedia and the median eminence-pituitary stalk region remain avascular throughout embryonic life.     

Localization of neurophysin in the neurosecretory elements of the hypothalamus and neurohypophysis of the normal and osmotically stimulated guinea-pig as demonstrated by immunofluorescence histochemical techniques

Summary Antisera to porcine neurophysin-II and ovine neurophysin-III were used to localize neurophysin-like material in the hypothalamus and neurohypophysis of guinea-pigs with an immunofluorescence technique. Although the guinea-pig appears to have only one major neurophysin it was found to be localized in both of the bilateral magnocellular nuclei. Neurophysin-like material was also present in extreme rostral portions of the hypothalamus, in cells lying between the third ventricle and the supraoptic nucleus and in a cluster of cells dorsomedial to the fornix. Immunofluorescence was observed in neurosecretory fibres that followed pathways previously characterized with classical histological stains for neurosecretory material.The immunofluorescence in the hypothalamic elements of a normal guinea-pig was not greatly different from that in fluorescent structures present in an animal that had been severely dehydrated. In contrast, there was a marked depletion of neurophysin from the posterior pituitary gland of the dehydrated guinea-pig. The lack of graded fluorescence in the hypothalamus of the dehydrated animal is discussed.This work was financed by a grant from the New Zealand Medical Research Council and the Auckland Medical Research Foundation.