Morphological studies on neuroglia

Immunohistochemical studies with the use of the peroxidase-antiperoxidase (PAP) method revealed that "amoeboid microglial cells", in the brains of neonatal rats and "brain macrophages" in lesioned brains of adult rats react positively to an antiserum raised against macrophages. In brains of neonatal rats, "amoeboid microglial cells" stained by means of the PAP-method were observed in the corpus callosum, internal capsule, dorso-lateral region of the thalamus, subventricular zone of the lateral ventricle, and the subependymal layer of the ventricular system. These cellular elements were not detected in brains of rats aged 21 days or older. Resting microglial cells displaying a typical ramified structure were not specifically stained. Cells reacting positively to the macrophage antiserum appeared (i) in the cerebral cortex of adult rats following placement of a stab wound, or (ii) in the hippocampal formation after kainic acid-induced lesions; in the damaged areas immunoreactive cells exhibited the typical features of "brain macrophages". "Brain macrophages" and "amoeboid microglial cells" are considered to belong to the class of exudate macrophages derived from blood monocytes. Thus, elements of hematogenous origin do exist in the intact brain parenchyma of neonatal rats and in lesioned brains of adult rats. The relationship between brain macrophages and resting microglial cells is discussed.     

Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Neurotoxicity of Glucocorticoids in the Primate Brain

Severe and prolonged physical and psychological stress is known to cause brain damage; long-term torture victims in prison bare later developed psychiatric disorders and cerebral cortical atrophy observed in CT scans (Jensen, Genefke, Hyldebrandt, Pedersen, Petersen, and Weile. 1982). In nonhuman primates, we observed degeneration and depletion of the hippocampal neurons in African green monkeys that had been severely abused by cagemates and died with complications of multiple gastric ulcers and adrenal cortical hyperplasia (Uno, Tarara, Else, Suleman and Sapolsky, 1989). In our previous studies the administration of dexamethasone (DEX) (5 mg/kg) to pregnant rhesus monkeys at 132 to 133 days of gestation induced degeneration and depletion of the hippocampal pyramidal and dentate granular neurons in the brains of 135-gestation-day fetuses, and these changes were retained in the brains of fetuses at near term, 165 days of gestation (Uno, Lohmiller, Thieme, Kemnitz, Engle, Roecker, and Farrell, 1990). We also found that implantation of a cortisol pellet in the vicinity of the hippocampus in adult vervet monkeys induced degeneration of the CA3 pyramidal neurons and their dendritic branches (Sapolsky, Uno, Rebert, and Finch, 1990). Thus, hippocampal pyramidal neurons containing a high concentration of glucocorticoid receptors appear to be highly vulnerable to either hypercortisolemia caused by severe stress or to exposure to exogenous glucocorticoids. To study the long-term postnatal sequelae of prenatal brain damage, eight rhesus monkeys were treated with either DEX (5 mg/kg), 5 animals, or vehicle, 3 animals, at 132 to 133 days of gestation. After natural birth, all animals lived with their mothers for 1 year. At 9 months of age, we found that DEX-treated animals had significantly high plasma cortisol at both base and post stress (isolation) levels compared to age-matched vehicle-treated animals. Magnetic resonance images (MRI) of the brain at 20 months of age showed an approximately 30% reduction in size and segmental volumes of the hippocampus in DEX-treated compared to vehicle-treated animals. Measurements of whole brain volume by MRI showed no significant differences between DEX and vehicle groups. Prenatal administration of a potent glucocorticoid (DEX) induced an irreversible deficiency of the hippocampal neurons and high plasma cortisol at the circadian baseline and post-stress levels in juvenile rhesus monkeys. These results suggest that the hippocampus mediates negative feedback of cortisol release; a lack or deficiency of the hippocampal neurons attenuates this feedback resulting in hypercortisolemia. The hippocampal deficiency in rhesus monkeys induced by prenatal administration of DEX appears to be a good model for neuroendocrinological dysfunctions and hippocampal development in human juveniles whose mothers were exposed to severe stress or received a high dose of glucocorticosteroids during pregnancy.     

Mutant α-synuclein and aging reduce neurogenesis in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

Neurogenesis, the production of new neurons from less differentiated precursor cells, normally occurs in adult brains in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Neurogenesis declines with aging. In previous studies, neurogenesis was stimulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) in young animals. In this study, we examined the effect of acute MPTP administration and mutant α-synuclein A53T on neurogenesis and migration of newborn neurons in the aged (23-month) vs. young (2-month) rodent brain. Cell proliferation and neurogenesis were assessed via bromodeoxyuridine labeling and immunostaining for cell type-specific markers. In the aged brain, neural precursor cells in the rostral SVZ retained the capacity for proliferation and migration in response to MPTP-induced Parkinsonism, although the response is less robust than in younger animals. Furthermore, in transgenic mice that overexpress mutant α-synuclein (A53T), brains examined day 21 after MPTP administration showed markedly decreased olfactory bulb and substantia nigra neurogenesis. Our data suggest that in addition to aging effects associated with decline in the number of newly generated cells, mutant α-synuclein reduces MPTP-induced neurogenesis. This could provide a novel therapeutic target for chronic brain repair in this condition.     

Comparison of NADPH diaphorase activity in the brains of hamsters infected with scrapie strains 139H or 263K or with normal hamster brain homogenate

Previous studies showed that the histopathological changes found in the brains of scrapie-infected animals included amyloid plaque formation, vacuolation, gliosis and neuronal and neurite degeneration. There were differences in the histopathological findings as a function of the scrapie strain-host combination. NADPH-diaphorase (NADPH-d) has been shown to be a selective histochemical marker for neurons containing nitric oxide (NO) synthase. Neuronal cell damage caused by NOS in brain has been reported to be associated with many neurodegenerative diseases. In this study, we used NADPH-d histostaining to investigate changes in the NOS system in brains of 139H- and 263K-infected hamsters and compared the results to normal hamster brain (NHB) injected animals. We observed that some of the NADPH-d histostaining neurons in the cortex of scrapie-infected hamsters appeared to be atrophic: the neurons were smaller and had fewer neurites. The NADPH-d histostaining intensity of neurons or astrocytes in septum, thalamus, hypothalamus and amygdala of 139H- and 263K-infected hamsters was greater than in control hamsters. Astrocytes in the thalamus, hypothalamus and lower part of the cortex (layers 4 to 6) in 263K-infected hamsters were more intensely stained for NADPH-d than in either 139H-infected hamsters or controls. Our results suggest that changes in NADPH-d system might play a role in the diversity of scrapie induced neurodegenerative changes.     

Evaporative light scattering detection of pyrrolizidine alkaloids

A reverse-phase high-performance liquid chromatography method utilizing evaporative light scattering detection (ELSD) has been developed for the simultaneous detection of hepatotoxic pyrrolizidine alkaloids with and without chromophores, namely, riddelliine, riddelliine N-oxide, senecionine, senecionine N-oxide, seneciphylline, retrorsine, integerrimine, lasiocarpine and heliotrine. Pyrrolizidine alkaloids were detected in five plant extracts (Senecio spartioides, S. douglasii var. longilobus, S. jacobaea, S. intergerrimus var. exaltatus and Symphytum officinale). The detection of heliotrine (which does not contain a chromophore) was much improved by ELSD compared with photodiode array detection.     

Presenilin-2 in the cynomolgus monkey brain: investigation of age-related changes

Localization of presenilin-2 (PS-2), a transmembrane protein implicated in early onset familial Alzheimers disease, was examined in the brains of 30 cynomolgus monkeys aged 4 to 36 years. Anti-PS-2 antibody N20, which recognizes PS-2 amino acid residues 2–20, and anti-PS-2 antibody C20, which recognizes PS-2 amino acid residues 535–554, stained mainly the cytoplasm of large pyramidal neurons and large neurites. This finding was also confirmed by double immunohistochemical investigations using N20 or C20 and anti-NeuN antibody. In the brain of the oldest monkey, swollen neurites containing senile plaques were immunostained with C20, but not with N20. Western blot analyses of microsomal fractions isolated from the brains of three adult monkeys revealed that much less PS-2 was present compared to presenilin-1 (PS-1). Age-related assessment of PS-2 in brain homogenates from young and adult monkeys showed that PS-2 levels and PS-2 subcellular localization were unchanged with increasing age. Because PS-2 expression was much less robust than that of PS-1, we conclude that PS-2 mainly localizes to large neurons and does not show so drastic age-related changes as PS-1.     

Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

Caspase-3 immunoreactivity in different cortical areas of young and aging macaque (Macaca mulatta) monkeys

It has been shown that cytochrome-c-dependent caspase-3 activation is significantly elevated in the aging macaque brain. To assess the underlying age-related changes in the cellular distribution of caspase-3, we have examined the motor cortex, cerebellum and hippocampus of young (4-year-old, n = 4) and old (20-year-old, n = 4)rhesus monkeys by immunohistochemistry. Western blot analyses of brain homogenate showed that the antibody reacted only with inactive 32-kDa procaspase and its active 20- and 17-kDa subunits, formed after granzyme B exposure. In the motor cortex, pyramidal cells of layers III and V were moderately labeled; the underlying white matter contained weakly stained astrocytes. In the hippocampus, hilar neurons and pyramidal cells in CA3 showed the strongest immunoreaction, pyramidal cells in CA1 and granule cells of the dentate gyrus were also strongly labeled. In contrast, CA2 pyramidal cells were only weakly stained, and neurons of the molecular layer were unlabeled. Weak caspase-3 immunoreaction of CA2 neurons parallels known decreased susceptibility to apoptosis. In the cerebellar cortex, clusters of strongly labeled Purkinje cells were observed next to groups of weakly and unstained cells; granule cells were generally unstained. The brains of aging monkeys displayed a similar pattern of caspase-3 immunoreactivity. In neocortical layer V, however, scattered very strongly labeled pyramidal cells were regularly detected, which were not observed in younger animals. This clustering of caspase-3 indicates increased vulnerability of a subset of pyramidal cells in the aging brain.     

Visualization of erythrocytes in the zebrafish brain

We found that erythrocytes of zebrafish have cytoplasmic peroxidase activity. Blood in the zebrafish brain was visualized using a standard peroxidase staining method after formaldehyde fixation. The erythrocytes in the brain were heavily stained, but neurons and glias were not stained at all. This easy method enables the distribution of erythrocytes in the whole brain to be determined, and enables the actual number of erythrocytes in each area in the brain to be calculated. The paths of major, thick blood vessels in zebrafish brain are similar to those in higher vertebrates, however, the distribution of thin blood vessels is different. We also found that the erythrocytes were unevenly distributed in the brain. For example, the density of erythrocytes in the surface layer of the tectum was more than 30-fold higher than in the deeper granular layer. Very few erythrocytes were found in bundles of axons like cranial nerves and the medial longitudinal fascicle. In general, fewer erythrocytes were found in areas near the ventricle, whereas many more were found closer to the surface of the brain. The distribution of erythrocytes in the brains of sleeping, awake and actively moving fish were compared. In the brains of sleeping fish, most of the erythrocytes were present in large vessels. This was not observed in brains of awake or actively moving fish. We found that the blood supply to motor neurons in the ventral horn of the spinal cord increased during active movement compared to that in awake or sleeping fish.     

Microtubule assembly in developing mammalian brain.

Microtubule protein was measured in mouse brain homogenates by quantitative colchicine binding. Neonatal animals contained more than twice the amount of brain tubulin as adult mice. The percentage of colchicine-binding protein which was polymerized was determined by extracting brain at room temperature into a medium designed to stabilize intact microtubules. Under identical conditions and tubulin concentrations, neonatal brain tubulin (colchicine-binding activity) had a greater proportion of the total extracted in an apparently polymerized state (pelletable by centrifugation) than did adult brain. A slight variation in the ratio of assembled to unassembled tubulin was observed with varying protein concentration (volume of extract), indicating that the values obtained may not reflect exactly the in vivo situation, because a rapid equilibration takes place upon homogenization. At all protein concentrations, the neonatal brain extracts contained a significantly greater proportion of assembled tubulin than did adult brain. This proportion began to fall at 5 days postnatal and reached the adult level at 30 days. The tubulin assembled/not assembled ratios were not altered by addition of nucleoside triphosphates, additional EGTA, or sulfhydryl protecting agents, and did not vary with preparation times of 30–90 min. The colchicine-binding reaction and decay of colchicine-binding activity with time were similar in extracts of different aged mouse brains, with neonatal slightly more stable than adult. Pools of tubulin from any age which were soluble at room temperature (unpolymerized) could not repolymerize well in vitro when incubated with GTP at 37 °C, whereas pools of tubulin which were sedimentable at room temperature (polymerized) could be redissolved at 0 °C and readily reassembled at 37 °C. The neonatal extract tubulin was thus more polymerization competent than the adult extracts; this correlates with a greater proportion of assembled tubulin in extracts at room temperature and possibly in vivo.     

Phospholipid metabolism in neuronal and glial cells during aging

The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.     

In vitro inhibition of the corpora allata by a larval brain factor in the cockroach Diploptera punctata

The inhibitory activity of extracts of brains from final instar male and female larvae on release of newly synthesized juvenile hormone (JH) was examined, as was the responsiveness of corpora allata (CA) from males and females to brain extract. Aqueous extracts of protocerebra from day 0 and day 14 final instar larvae (in which the CA are active and inactive, respectively) were tested on CA from adult females in vitro. For comparison, protocerebra from day 0 adult males and females were also tested. Dose responses were similar for protocerebra from all animals tested. In each case a dose of 1–2 protocerebral equivalents was required for maximal response.CA from penultimate instar females were inhibited to a greater extent by brain extract than those of males. The response of CA from final instar larvae to brain extract declined in the first few days of the stadium as the ability of the glands to biosynthesize JH declined.     

Responses of olfactory receptor neurons in Utetheisa ornatrix to gender-specific odors

We recorded the electrophysiological responses of individual olfactory receptor neurons in sensilla trichodea on the antennae of adult arctiid moths, Utetheisa ornatrix, to stimulation with volatiles associated with both sexes. All trichoid sensilla contain at least two receptor neurons, each with distinct action potential amplitudes and waveforms, that respond dichotomously to male and female odors. Although, neither female neuron responds to extracts of coremata or the male-produced pheromone hydroxydanaidal, they do respond in a gender-specific manner to the volatiles emanating from whole pupae, hemolymph, thoracic froth, and adult animals of several ages. Thoracic froth, which contains pyrrolizidine alkaloids, is thought to play a role in defense. Froth from moths reared on diets, with or without added pyrrolizidine alkaloids, were equally effective in eliciting gender-specific patterns of response. Male trichoid receptor neurons respond to these same materials with similar patterns of activation. These receptor neurons provide information about substances, which we have termed “gender odors,” that are persistently emitted by nearby animals. These substances do not appear to be the same as those already known to be involved in defense or the sexual dialog between individuals. Accepted: 16 March 2000     

Immunocytochemistry and heterogeneity of rat brain vimentin

In unfixed cryostat sections of the brains of early postnatal and adult rats, we screened for cells containing vimentin-positive intermediate filaments (VI+-IFs) by applying a panel of four monoclonal antibodies (Mabs VI-01, VI-02, VI-05 and VI-5B3) using indirect immunofluorescence. All of the Mabs stained VI+-IFs in the stromal part of the choroid plexus, in endothelial cells of blood vessels and in meninges in both adult and immature brains, although with varying strength (VI-5B3 and VI-01 stained more strongly than VI-05 and VI-02). In the brain parenchyma of adults, intense staining was mainly localized in ventricular ependymal cells (VI-5B3/VI-01 greater than VI-02/VI-05) and fibrous astrocyte-like cells (FAs). In the immature brain, the ependymal cells were activated in appearance, with evidence of cell enlargement, greater spreading of VI+-IFs within the cytoplasm and more pronounced VI+ cytoplasmic protrusions into the brain parenchyma. VI+-FAs were found near the ependymal and meningeal borders as well as in the white matter tracts of adult brain (VI-5B3/VI-01 greater than VI-05 greater than VI-02). In immature animals, VI+-FAs were less frequently encountered in the forebrain regions, except in and near the subepenydmal layer (in the adjacent parenchyma) as well as in submeningeal layers. Weaker staining was usually elicited by Mabs VI-02 and VI-05. In the cerebellum, Bergmann cell fibers were stained in both age groups. In adults, the most intense fluorescence usually occurred in segments close to the pia (VI-5B3/VI-01 greater than VI-05 greater than VI-02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of nestin by neural cells in the adult rat and human brain

Neurons and glial cells in the developing brain arise from neural progenitor cells (NPCs). Nestin, an intermediate filament protein, is thought to be expressed exclusively by NPCs in the normal brain, and is replaced by the expression of proteins specific for neurons or glia in differentiated cells. Nestin expressing NPCs are found in the adult brain in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. While significant attention has been paid to studying NPCs in the SVZ and SGZ in the adult brain, relatively little attention has been paid to determining whether nestin-expressing neural cells (NECs) exist outside of the SVZ and SGZ. We therefore stained sections immunocytochemically from the adult rat and human brain for NECs, observed four distinct classes of these cells, and present here the first comprehensive report on these cells. Class I cells are among the smallest neural cells in the brain and are widely distributed. Class II cells are located in the walls of the aqueduct and third ventricle. Class IV cells are found throughout the forebrain and typically reside immediately adjacent to a neuron. Class III cells are observed only in the basal forebrain and closely related areas such as the hippocampus and corpus striatum. Class III cells resemble neurons structurally and co-express markers associated exclusively with neurons. Cell proliferation experiments demonstrate that Class III cells are not recently born. Instead, these cells appear to be mature neurons in the adult brain that express nestin. Neurons that express nestin are not supposed to exist in the brain at any stage of development. That these unique neurons are found only in brain regions involved in higher order cognitive function suggests that they may be remodeling their cytoskeleton in supporting the neural plasticity required for these functions.     

Open field stress and neurons containing calcium-binding proteins in the piriform cortex of the rat.

In the present study we wanted to check whether the expression of the c-Fos protein (the marker of cellular activity) appears in cells containing calcium-binding proteins (CaBPs) in animals exposed to the open field test. Eight adult Wistar rats were examined. In the first step the open field test was applied throughout 10 minutes. After perfusional fixation brains were frozen and cut on the cryostat in the coronal plane and stained with the standard immunohistochemical method. Sections were double stained for c-Fos and CaBPs: parvalbumin (PV), calbindin (CB), calretinin (CR). c-Fos positive cells were localized predominantly in layers II and III of the piriform cortex (PC). The double labeling study showed that neurons containing CaBPs are rarely c-Fos-immunoreactive. Often PV-positive and CB-positive fibers surround c-Fos-positive neurons in layers II and III in a form of a basket. It seems that cells containing CaBPs are not directly involved in the response to aversive stimuli but cells containing those calcium-binding proteins might influence directly c-Fos positive neurons of PC.     

Protective and regenerative response endogenously induced in the ischemic brain

Neuronal cells are highly vulnerable to ischemic insult. Because adult neurons are highly differentiated and cannot self-propagate, loss of neurons often results in functional deficits in mammalian brains. However, it has recently been shown that neurons and neuronal circuits exhibit protective and regenerative responses in a rodent model of experimental ischemia. At first, neurons respond by producing several protective proteins such as heat shock proteins (HSPs) after sublethal ischemia and then acquire tolerance against a subsequent ischemic insult (ischemic tolerance). Once neurons suffer irreversible injury, two repair processes, neurogenesis and synaptogenesis, are endogenously induced. Neuronal stem and (or) progenitor cells can proliferate in two brain areas in adult animals: the subventricular zone and the subgranular zone in the dentate gyrus. After ischemic insult, these stem (progenitor) cells proliferate and differentiate into neurons in the dentate gyrus of the hippocampus. Reactive synaptogenesis has been also observed in the injured brain following a period of long-term infarction, but it is unclear if it can compensate for disconnected circuits. Understanding the molecular mechanism underlying these protective and regenerative responses will be important in developing a new strategy for aimed at the augmentation of resistance against ischemic insult and the replacement of injured neurons and neuronal circuits.     

Quinolizidine alkaloid profiles of Lupinus varius orientalis, L. albus albus, L. hartwegii, and L. densiflorus

Alkaloid profiles of two Lupinus species growing naturally in Egypt (L. albus albus [synonym L. termis], L. varius orientalis) in addition to two New World species (L. hartwegii, L. densiflorus) which were cultivated in Egypt were studied by capillary GLC and GLC-mass spectrometry with respect to quinolizidine alkaloids. Altogether 44 quinolizidine, bipiperidyl and proto-indole alkaloids were identified; 29 in L. albus, 13 in L. varius orientalis, 15 in L. hartwegii, 6 in L. densiflorus. Some of these alkaloids were identified for the first time in these plants. The alkaloidal patterns of various plant organs (leaves, flowers, stems, roots, pods and seeds) are documented. Screening for antimicrobial activity of these plant extracts demonstrated substantial activity against Candida albicans, Aspergillus flavus and Bacillus subtilis.